Enzymes for trimming of glycoproteins

ABSTRACT

The invention concerns fusion proteins, wherein two endoglycosidases are fused, possibly via a linker. The fusion enzymes according to the invention have structure (1): EndoX-(L) p -EndoY (1), wherein EndoX is an endoglycosidase, EndoY is an endoglycosidase distinct from EndoX, L is a linker and p is 0 or 1. Such fusion enzymes capable of trimming glycoproteins comprising at least two distinct glycoforms in a single step. The invention further concerns the use of the fusion enzyme according to the invention for trimming glycoproteins. In another aspect, the invention relates to the process of production of the fusion enzyme. In a further aspect, the inventions concerns a process for trimming glycoproteins, comprising trimming the glycoprotein with a fusion enzyme according to the invention, to obtain a trimmed glycoprotein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Phase of International PatentApplication No. PCT/EP2017/052792, filed Feb. 8, 2017, published on Aug.17, 2017 as WO 2017/137459 A1, which claims priority to European PatentApplication No. 16154712.0, filed Feb. 8, 2016, and claims priority toEuropean Patent Application No. 16154739.3, filed Feb. 8, 2016, andclaims priority to European Patent Application No. 16173595.6 filed Jun.8, 2016, and claims priority to European Patent Application No.16173599.8 filed Jun. 8, 2016, and claims priority to European PatentApplication No. 16206867.0 filed Dec. 23, 2016. The contents of theseapplications are herein incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 7, 2018, isnamed 069818-4030_2018-08-07_Sequence_Listing.txt and is 157 KB.

FIELD OF THE INVENTION

The present invention is in the field of enzymatic hydrolysis ofoligosaccharides, more in particular to the trimming of glycoproteins.The invention relates to improved enzymes for such trimming to liberatethe core GlcNAc and to a process for trimming of glycoproteins using theenzymes according to the invention.

BACKGROUND OF THE INVENTION

Glycoproteins exist in many glycosylated variants, or glycoforms, whichcan differ substantially in their biochemical properties and(biological) functions. Glycans are structurally diverse, incorporatinga wide range of monosaccharide residues and glycosidic linkages.

Many therapeutic proteins are glycoproteins, and although some arepurified from natural sources, the majority are recombinantly expressed.The choice of expression system heavily influences the glycosylation.There have been notable efforts in controlling the glycosylation ofglycoprotein production systems motivated by the impact on in vivofunctionality. For example, monoclonal antibodies with engineeredglycosylation display enhanced pharmacokinetics and effector function.Glycopeptides offer intriguing possibilities in the development ofanticancer vaccines given their ability to stimulate both humoral andcellular immunity. Additionally, the HIV glycan shield is an effectivetarget for antibody neutralization and an emerging target for vaccinedesign.

On the other hand, removal of N-glycans from glycoproteins providescomplementary therapeutic opportunities. Deglycosylation of IgGsignificantly decreases binding of antibodies to Fc-gamma receptors,thereby avoiding aspecific uptake of antibodies by e.g. macrophages ormegakaryocytes, which may lead to thrombocytopenia. The latterbiological phenomenon is responsible for the dose-limiting toxicity(DLT) of Kadcyla®, an antibody-drug-conjugate to treat HER2-upregulatedbreast cancer. Selective deglycosylation of antibodies in vivo affordsopportunities to treat patients with antibody-mediated autoimmunity.Removal of high-mannose glycoform in a recombinant therapeuticglycoprotein may be beneficial, since high-mannose glycoforms are knownto compromise therapeutic efficacy by aspecific uptake by endogenousmannose receptors and leading to rapid clearance, as for exampledescribed by Gorovits and Krinos-Fiorotti, Cancer Immunol. Immunother.2013, 62, 217-223 and Goetze et al, Glycobiology 2011, 21, 949-959 (bothincorporated by reference). In addition, Van de Bovenkamp et al, J.Immunol. 2016, 196, 1435-1441 (incorporated by reference) describe howhigh-mannose glycans can influence immunity. It was described by Reuschand Tejada, Glycobiology 2015, 25, 1325-1334 (incorporated byreference), that inappropriate glycosylation in monoclonal antibodiescould contribute to ineffective production from expressed Ig genes. Insome cases, a carbohydrate addition sequence generated by either Vregion rearrangement or somatic hypermutation may result in an antibodythat cannot be properly folded and secreted, as described by Gala andMorrison, J. Immunol. 2004, 172, 5489-5494 (incorporated by reference).

An additional advantage of deglycosylated therapeutic proteins is themuch facilitated batch-to-batch consistency and significantly improvedhomogeneity, which is highly advantageous for regulatory approval.

A highly useful and straightforward approach to obtain deglycosylatedrecombinant proteins, thereby offering a route to improving the efficacyof therapeutic antibodies and other N-glycoproteins, is by enzymaticremoval of glycans. Fortuitously, endoglycosidases have been discoveredthat are able to cleave N-glycans, which offers the possibility ofselective removal from a recombinant glycoprotein. Endoglycosidases havefurther found use in the preparation of conjugates from glycoproteins,by selectively liberating the core GlcNAc moieties upon trimming,followed by bioconjugation. Another field of use of endoglycosidases ismass spectrometry, one of the key analytical tools for characterizing(therapeutic) proteins, including glycoproteins and monoclonalantibodies in particular. By enzymatic cleavage of the complex andheterogeneous glycan from the protein, mass spectrometric analysis issignificantly facilitated.

Bioconjugation is the process of linking two or more molecules, of whichat least one is a biomolecule and the other molecule(s) may be referredto as “target molecule” or “molecule of interest”. Many differentcompounds have been found useful to be conjugated to glycoproteins. Forexample, the modulation of protein structure and function by covalentmodification with a chemical probe for detection and/or isolation hasevolved as a powerful tool in proteome-based research and biomedicalapplications. Fluorescent or affinity tagging of proteins is key tostudying the trafficking of proteins in their native habitat, andvaccines based on protein-carbohydrate conjugates have gained prominencein the fight against HIV, cancer, malaria and pathogenic bacteria.PEGylation of proteins or attachment of a protein to serum albumin areuseful strategies to enhance the pharmacokinetic profile by reducingclearance rates, whereas functionalization of a carrier protein such asa monoclonal antibody with a toxic payload is a promising strategy inthe targeted treatment of disease (in particular cancer).

In general, two strategic concepts can be recognized in the field ofbioconjugation: (a) conjugation based on a native functional group (inother words: a functional group already present in the biomolecule ofinterest, such as for example a thiol, an amine, an alcohol or ahydroxyphenol unit) or (b) a two-stage process involving engineering ofone (or more) unique reactive groups into a biomolecule prior to theactual conjugation process.

The first approach typically involves a reactive amino acid side-chainin a protein (e.g. cysteine or lysine), or a functional group in aglycan (e.g. amine, aldehyde) or nucleic acid (e.g. purine or pyrimidinefunctionality or alcohol). As summarized inter alia in G. T. Hermanson,“Bioconjugate Techniques”, Elsevier, 3^(rd) Ed. 2013, incorporated byreference. Most prominently, cysteine-maleimide conjugation stands outfor protein conjugation by virtue of its high reaction rate andchemoselectivity. However, when no free cysteine is available forconjugation, as in many proteins, other methods are often required, eachsuffering from its own shortcomings especially in terms ofsite-specificity. Moreover, a general disadvantage of protein conjugatesobtained via alkylation with maleimides is that in general the resultingsuccinimide conjugates can be unstable due to the reverse of alkylation,i.e. a retro-Michael reaction.

An elegant and broadly applicable solution for bioconjugation involvesthe two-stage approach. Although more laborious, two-stage conjugationvia engineered functionality typically leads to higher selectivity(site-specificity) than conjugation on a natural functionality. Besidesthat, full stability can be achieved by proper choice of construct.Typical examples of a functional group that may be imparted onto thebiomolecule include (strained) alkyne, (strained) alkene, norbornene,tetrazine, azide, phosphine, nitrile oxide, nitrone, nitrile imine,diazo compound, carbonyl compound, (O-alkyl)hydroxylamine and hydrazine,which may be achieved by either chemical or molecular biology approach.Each of the above functional groups is known to have at least onereaction partner, in many cases involving complete mutual reactivity.For example, cyclooctynes react selectively and exclusively with1,3-dipoles, strained alkenes with tetrazines and phosphines withazides, leading to fully stable covalent bonds.

An efficient route towards the introduction of engineeredfunctionalities such as azides into specifically glycoproteins is viaselective functionalization of the glycans present on the glycoprotein.All recombinant antibodies, generated in mammalian host systems, containthe conserved N-glycosylation site on the asparagine residue at or closeto position 297. These glycans are always formed as a complex mixture ofisoforms, see e.g. FIGS. 1 and 2, consisting of a highly heterogeneousmixture of complex, hybrid and high-mannose glycans. Trimming of theseglycans by an endoglycosidase leaves only the core GlcNAc moiety(attached to N297), optionally fucosylated at the 6-OH group. Theliberated core GlcNAc provides a suitable anchor point for targetmolecules, providing a product with a much higher homogeneity incomparison to products obtained by conjugation to terminal sugarmoieties present in the original glycan structure. A downside of thisapproach, however, is that different glycans may require differentendoglycosidases, each with their own optimal conditions, such thatmultiple enzymatic treatments may be required for proper and completetrimming of the glycoprotein. For example, EndoH is known to trimhigh-mannose and hybrid glycoforms, but not complex type glycans, whileEndoS is able to trim complex type glycans and to some extent hybridglycan, but not high-mannose forms. EndoF2 is able to trim complexglycans (but not hybrid), while endoF3 can only trim complex glycansthat are also 1,6-fucosylated. Another endoglycosidase, EndoD is able tohydrolyze Mans (M5) glycan only. An overview of specific activities ofdifferent endoglycosidases is disclosed in Freeze et al. in Curr.Protoc. Mol. Biol., 2010, 89:17.13A.1-17, incorporated by referenceherein.

Yamamoto et al. disclose in Glycoconjugate J. 2005, 22, 35-42,incorporated by reference herein, a chimeric construct of EndoD andEndoBH, which was completely inactive. The chimeric construct wasdesigned to investigate the homology of both endoglycosidases intrimming of glycans. In the context of glycoprotein conjugation, WO2007/095506 and WO 2008/029281 disclose that trimming of the glycan cantake place with EndoH, thereby hydrolysing a GlcNAc-GlcNAc glycosidicbond and liberating a GlcNAc for enzymatic introduction of GaINAz. VanGeel et al. disclose in Bioconjugate Chem. 2015, 26, 2233, incorporatedby reference herein, that transfer of a range of azido-modifiedgalactose moieties to the core GlcNAc residue of a monoclonalantibodies, obtained by trimming with an endoglycosidase, followed byattachment of a toxic payload by means of copper-free click chemistry,is an efficient method to obtain antibody-drug conjugates with ademonstrated improved efficacy and safety profile versus marketed drugKadcyla®.

As a product of recombinant DNA technology, fusion proteins have beendeveloped as a class of novel biomolecules with multi-functionalproperties. By genetically fusing two or more proteins or proteindomains together, a fusion protein product is generated that may displaysimilar or distinctly different functions as those of the componentmoieties. Fusion proteins have found applications in purificationstrategies, immobilization, imaging, and biopharmaceuticals. Forexample, many protein drugs are fused to Fc domains of antibodies, suchas Fc-immunoglobulin G1 (Fc-IgG1), or to carrier proteins such as humanserum albumin (HSA) or transferrin (Tf) to extend their plasmahalf-lives and to achieve enhanced therapeutic effects. Several fusionproteins drugs including Enbrel® (tumour necrosis factor/Fc-IgG1),Ontak® (interleukin-2/diphtheria toxin), Orencia® (cytotoxicT-lymphocyte antigen-4/Fc-IgG1), Amevive® (leukocyte functionantigen-3/Fc-IgG1), Arcalyst® (interleukin-1 receptor extracellulardomain/Fc-IgG1), and Nplate® (thrombopoietin/Fc-IgG1) have been approvedby the FDA for therapeutic application. One relevant example of a fusionprotein of an endoglycosidase can be found in Warren et al., Prot. Eng.Design Select. 2005, 18, 497-501 (incorporated by reference), disclosinga fusion of carbohydrate binding domain (CBM) to EndoF1 or PNGaseF.

The successful construction of a recombinant fusion protein requires thecomponent proteins, but also the linkers may play an important role.Linkers may be short or long, flexible or rigid, and of cleavable ornon-cleavable nature. In some cases, the linker may increase stabilityor folding, improve expression or biological activity, or alterpharmacokinetics. Typical nature of linkers known in the art areoligomers of glycine, e.g. G₈, oligomers of GGGGS, oligomers of EAAAKand variants thereof. A recent overview of linkers for fusion proteinscan be found in Chen et al., Adv. Drug Deliv. Rev. 2013, 65, 1357-1369,incorporated herein by reference.

SUMMARY OF THE INVENTION

The invention concerns fusion proteins, wherein two endoglycosidases arefused, possibly via a linker. The fusion enzymes according to theinvention are conveniently capable of trimming glycoproteins comprisingat least two distinct glycoforms in a single step. All glycans ofglycoproteins, which cannot be removed by a single conventional enzyme,are completely trimmed to liberate the core GlcNAc by the fusion enzymeaccording to the invention. Surprisingly, both activities of the fusionenzyme function smoothly at the optimal pH of one of theendoglycosidases, while the other endoglycosidase may normally require adifferent pH to operate optimally. Moreover, it was found that theactivity of a particular endoglycosidase in a fusion protein can displaya higher trimming efficiency compared to the same endoglycosidase as asingle enzyme. The invention further concerns the use of the fusionenzyme according to the invention for trimming glycoproteins. In anotheraspect, the invention relates to the process of production of the fusionenzyme. In a further aspect, the inventions concerns a process fortrimming glycoproteins, comprising trimming the glycoprotein with afusion enzyme according to the invention, to obtain a trimmedglycoprotein.

DESCRIPTION OF THE FIGURES

FIG. 1 shows exemplary glycans of high-mannose type (Mans (M3), Mans(M5) and Mans (M9)), complex type (biantennary), bisected type,triantennary and tetraantennary type.

FIG. 2 shows an antibody comprising a glycan on each heavy chain. Themost typical complex glycosylation patterns of a recombinant antibodyare G1F, G0F and G1. Some mannose-type glycosylation may also be present(M5) and in some cases a hybrid type glycan (e.g. M5G1).

FIG. 3 shows the results of trimming of high-mannose glycoprotein RNAseBin Tris pH 7.5 by endoglycosidases EndoS, EndoS2 and EndoSH.Concentration series: 0.025, 0.125, 0.125 (duplo), and 0.25 mg/mL. Upperband=intact RNaseB, lower band=trimmed RNaseB.

FIG. 4 shows a plot of percentage conversion (trimming) of cAC10 at pH 6by endoglycosidases EndoS, EndoS2 and EndoSH as obtained in Example 14.Trendline for EndoSH: y=2.0784x+1.027 (R²=0.9982). Trendline for EndoS:y=2.0103x (R²=0.9838). Trendline for EndoS2: y=1.0297x−0.1622(R²=0.9998).

FIG. 5 shows a plot of percentage conversion (trimming) high-mannosetrastuzumab at pH 6 by endoglycosidases EndoS, EndoS2 and EndoSH asobtained in Example 15.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The verb “to comprise”, and its conjugations, as used in thisdescription and in the claims is used in its non-limiting sense to meanthat items following the word are included, but items not specificallymentioned are not excluded.

In addition, reference to an element by the indefinite article “a” or“an” does not exclude the possibility that more than one of the elementis present, unless the context clearly requires that there is one andonly one of the elements. The indefinite article “a” or “an” thususually means “at least one”.

The general term “sugar” is herein used to indicate a monosaccharide,for example glucose (Glc), galactose (Gal), mannose (Man) and fucose(Fuc). The term “sugar derivative” is herein used to indicate aderivative of a monosaccharide sugar, i.e. a monosaccharide sugarcomprising substituents and/or functional groups. Examples of a sugarderivative include amino sugars and sugar acids, e.g. glucosamine(GlcNH2), galactosamine (GalNH2) N-acetylglucosamine (GlcNAc),N-acetylgalactosamine (GalNAc), sialic acid (Sia) which is also referredto as N-acetylneuraminic acid (NeuNAc), and N-acetylmuramic acid(MurNAc), glucuronic acid (GlcA) and iduronic acid (IdoA).

The term “nucleotide” is herein used in its normal scientific meaning.The term “nucleotide” refers to a molecule that is composed of anucleobase, a five-carbon sugar (either ribose or 2-deoxyribose), andone, two or three phosphate groups. Without the phosphate group, thenucleobase and sugar compose a nucleoside. A nucleotide can thus also becalled a nucleoside monophosphate, a nucleoside diphosphate or anucleoside triphosphate. The nucleobase may be adenine, guanine,cytosine, uracil or thymine. Examples of a nucleotide include uridinediphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate(TDP), cytidine diphosphate (CDP) and cytidine monophosphate (CMP).

The term “protein” is herein used in its normal scientific meaning.Herein, polypeptides comprising about 10 or more amino acids areconsidered proteins. A protein may comprise natural, but also unnaturalamino acids.

Proteins and enzyme included mutants thereof. For example,“endoglycosidase” includes both native (wild-type) endoglycosidases andmutant endoglycosidases, as long as the endoglycosidase activity issubstantially maintained. A domain having an amino acid sequence that isdifferent from a wild-type amino acid sequence is herein referred to asa mutant domain. The mutation may e.g. comprise a single amino acidchange (a point mutation), but also multiple amino acid changes (e.g. of1 to 10, preferably of 1 to 6, more preferably of 1, 2, 3 or 4, evenmore preferably of 1 or 2 amino acids), or a deletion or insertion ofone or more (e.g. of 1 to 10, preferably of 1 to 6, such as 1, 2, 3 or4, preferably of 1 or 2) amino acids. Alternatively, larger deletions orinsertions can be applied to the enzyme. For example, truncatedendoglycosidase D (deletion of 599 amino acids from its C-terminalportion) has been found to retain its endoglycosidase activity (Yamamotoet al. in Glycoconjugate J. 2005, 22, 35-42). The skilled person isaware of the possibilities in this respect, and as long as theendoglycosidase activity is substantially retained the enzyme cancontain any type of mutation.

The term “glycoprotein” is herein used in its normal scientific meaningand refers to a protein comprising one or more monosaccharide oroligosaccharide chains (“glycans”) covalently bonded to the protein. Aglycan may be attached to a hydroxyl group on the protein(O-linked-glycan), e.g. to the hydroxyl group of serine, threonine,tyrosine, hydroxylysine or hydroxyproline, or to an amide function onthe protein (N-glycoprotein), e.g. asparagine or arginine, or to acarbon on the protein (C-glycoprotein), e.g. tryptophan. A glycoproteinmay comprise more than one glycan, may comprise a combination of one ormore monosaccharide and one or more oligosaccharide glycans, and maycomprise a combination of N-linked, O-linked and C-linked glycans. It isestimated that more than 50% of all proteins have some form ofglycosylation and therefore qualify as glycoprotein. Examples ofglycoproteins include PSMA (prostate-specific membrane antigen), CAL(candida antartica lipase), gp41, gp120, EPO (erythropoietin),antifreeze protein and antibodies.

The term “glycan” is herein used in its normal scientific meaning andrefers to a monosaccharide or oligosaccharide chain that is linked to aprotein. The term glycan thus refers to the carbohydrate-part of aglycoprotein. The glycan is attached to a protein via the C-1 carbon ofone sugar, which may be without further substitution (monosaccharide) ormay be further substituted at one or more of its hydroxyl groups(oligosaccharide). A naturally occurring glycan typically comprises 1 toabout 10 saccharide moieties. However, when a longer saccharide chain islinked to a protein, said saccharide chain is herein also considered aglycan. A glycan of a glycoprotein may be a monosaccharide. Typically, amonosaccharide glycan of a glycoprotein consists of a singleN-acetylglucosamine (GlcNAc), glucose (Glc), mannose (Man) or fucose(Fuc) covalently attached to the protein. A glycan may also be anoligosaccharide. An oligosaccharide chain of a glycoprotein may belinear or branched. In an oligosaccharide, the sugar that is directlyattached to the protein is called the core sugar. In an oligosaccharide,a sugar that is not directly attached to the protein and is attached toat least two other sugars is called an internal sugar. In anoligosaccharide, a sugar that is not directly attached to the proteinbut to a single other sugar, i.e. carrying no further sugar substituentsat one or more of its other hydroxyl groups, is called the terminalsugar. For the avoidance of doubt, there may exist multiple terminalsugars in an oligosaccharide of a glycoprotein, but only one core sugar.The end of an oligosaccharide that is directly attached to the proteinis called the reducing end of a glycan. The other end of theoligosaccharide is called the non-reducing end of a glycan. A glycan maybe an O-linked glycan, an N-linked glycan or a C-linked glycan. In anO-linked glycan a monosaccharide or oligosaccharide glycan is bonded toan O-atom in an amino acid of the protein, typically via a hydroxylgroup of serine (Ser) or threonine (Thr). For O-linked glycans, a widediversity of chains exist. Naturally occurring O-linked glycanstypically feature a serine or threonine-linked α-O-GalNAc moiety,further substituted with galactose, sialic acid and/or fucose. Thehydroxylated amino acid that carries the glycan substitution may be partof any amino acid sequence in the protein.

In an N-linked glycan a monosaccharide or oligosaccharide glycan isbonded to the protein via an N-atom in an amino acid of the protein,typically via an amide nitrogen in the side chain of asparagine (Asn) orarginine (Arg). For N-linked glycans, a wide diversity of glycans exist.Naturally occurring N-linked glycans feature an asparagine-linkedβ-N-GlcNAc moiety, in turn further substituted at its 4-OH withβ-GlcNAc, in turn further substituted at its 4-OH with β-Man, in turnfurther substituted at its 3-OH and 6-OH with α-Man, leading to theglycan pentasaccharide Man₃GlcNAc₂. The core GlcNAc moiety may befurther substituted at its 6-OH by α-Fuc. The pentasaccharideMan₃GlcNAc₂ is the common oligosaccharide scaffold of nearly allN-linked glycoproteins and may carry a wide variety of othersubstituents, including but not limited to Man, GlcNAc, Gal and sialicacid. The asparagine that is substituted with the glycan on itsside-chain is typically part of the sequence Asn-X-Y, with X being anyamino acid but proline and Y being either serine or threonine.

In a C-linked glycan a monosaccharide or oligosaccharide glycan isbonded to a C-atom in an amino acid of the protein, typically to aC-atom of tryptophan (Trp).

The term “antibody” is herein used in its normal scientific meaning. Anantibody is a protein generated by the immune system that is capable ofrecognizing and binding to a specific antigen. An antibody is an exampleof a glycoprotein. The term antibody herein is used in its broadestsense and specifically includes monoclonal antibodies, polyclonalantibodies, dimers, multimers, multispecific antibodies (e.g. bispecificantibodies), antibody fragments, and double and single chain antibodies.The term “antibody” is herein also meant to include human antibodies,humanized antibodies, chimeric antibodies and antibodies specificallybinding cancer antigen. The term “antibody” is meant to include wholeantibodies, but also antigen-binding fragments of an antibody, forexample an antibody Fab fragment, F(ab′)₂, Fv fragment or Fc fragmentfrom a cleaved antibody, a scFv-Fc fragment, a minibody, a diabody or ascFv. Furthermore, the term includes genetically engineered antibodiesand derivatives of an antibody. Antibodies, fragments of antibodies andgenetically engineered antibodies may be obtained by methods that areknown in the art. Typical examples of antibodies include, amongstothers, abciximab, rituximab, basiliximab, palivizumab, infliximab,trastuzumab, alemtuzumab, adalimumab, tositumomab-I131, cetuximab,ibrituximab tiuxetan, omalizumab, bevacizumab, natalizumab, ranibizumab,panitumumab, eculizumab, certolizumab pegol, golimumab, canakinumab,catumaxomab, ustekinumab, tocilizumab, ofatumumab, denosumab, belimumab,ipilimumab and brentuximab.

A “linker” is herein defined as a moiety that connects two or moreelements of a compound. For example, the fusion enzyme according to theinvention may contain a linker that connects the two endoglycosidaseunits. In the context of the fusion enzymes according to the presentinvention, linkers typically contain at least one amino acid and mostpreferably consist of one or more amino acids.

A “bioconjugate” is herein defined as a compound wherein a biomoleculeis covalently connected to a target molecule via a linker. Abioconjugate comprises one or more biomolecules and/or one or moretarget molecules. The linker may comprise one or more spacer moieties. Atarget molecule may be an active substance, a reporter molecule, apolymer, a solid surface, a hydrogel, a nanoparticle, a microparticle ora biomolecule.

The term “fusion enzyme” herein refers to an enzyme wherein the aminoacid sequences of two or more enzymes that originally belonged toseparate enzymes are joined together, optionally via a linker. Fusionenzymes are known in the art and may be created by the joining of two ormore genes that originally code for separate enzymes. Translation ofthis gene results in a single polypeptide with functional propertiesderived from each of the original enzymes.

Fusion Enzyme

In a first aspect, the invention concerns a fusion enzyme comprising twoendoglycosidases, optionally connected via a linker. The fusion enzymeaccording to the invention may be represented by structure (1):EndoX-(L)_(p)-EndoY  (1)

Herein, EndoX and EndoY are both individually an endoglycosidase, L is alinker and p is 0 or 1. In the context of the present invention, “fusionenzyme” may also be referred to as “fusion protein”. The fusion enzymeaccording to the invention is preferably an end-to-end fusion, eitherdirect or via a linker L.

Endoglycosidase

Endoglycosidase are known in the art as enzymes that cleaveoligosaccharides between two glycosidic bonds, as such releasing themfrom either glycoproteins, glycopeptides or glycolipids. Sucholigosaccharides are typically referred to as glycans. In the context ofthe present invention, “Endo” refers to endoglycosidase.Endoglycosidases hydrolyse the bond between two sugar units in anoligosaccharide or polysaccharide, but not between the core sugar unit,which is directly bound to the peptide part of a glycoprotein, and theamino acid it is connected to. Endoglycosidases typically hydrolyse thebond between the two core N-acetylglucosamine (GlcNAc) residues inN-linked glycans, thus leaving the core GlcNAc residue connected to thepeptide part of the glycoprotein.

In the context of the present invention, the term endoglycosidaseencompasses all members of the family of endoglycosidase that releasesoligosaccharides from glycoproteins, glycopeptides or glycolipids.Endoglycosidase may also cleave polysaccharide chains between residuesthat are not the terminal residue, although releasing oligosaccharidesfrom conjugated protein and lipid molecules is more common.

In the context of the present invention, the term endoglycosidaseencompasses both the native endoglycosidases or truncatedendoglycosidases and mutants thereof, as long as the endoglycosidaseactivity is substantially retained. In other words, the amino acidsequence of EndoX and EndoY may comprise a different amino acidssequence compared to the native endoglycosidase. In one embodiment, theamino acid sequence of EndoX and EndoY comprise a mutant. In oneembodiment, the amino acid sequence of EndoX and EndoY do not comprise amutant. In one embodiment, the amino acid sequence of EndoX and EndoYcomprise a truncated sequence. In one embodiment, the amino acidsequence of EndoX and EndoY do not comprise a truncated sequence. Whenlooking at the sequence of EndoX and EndoY individually, it is preferredthat each of EndoX and EndoY has at least 80% sequence identity with thecorresponding native amino acid sequence of the catalytic domain of theendoglycosidase, such as at least 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity with the corresponding native amino acid sequence. Mostpreferably, each of EndoX and EndoY has 100% sequence identity with thecorresponding amino acid sequence of the catalytic domain of theendoglycosidase. Alternatively or additionally, it is preferred thateach of EndoX and EndoY has at least 80% sequence similarity with thecorresponding native amino acid sequence, such as at least 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% sequence similarity with the corresponding native aminoacid sequence of the catalytic domain of the endoglycosidase. Mostpreferably, each of EndoX and EndoY has 100% sequence similarity withthe corresponding native amino acid sequence of the catalytic domain ofthe endoglycosidase.

Sequence identity and similarities can be readily calculated by knownmethods and/or computer program methods known in the art such as BLASTPpublicly available from NCBI and other sources (BLAST Manual, Altschul,S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J.Mol. Biol. 215:403-410 (1990), incorporated by reference.

Glycans that can be cleaved by glycosidases exist in various glycoforms,which are generally grouped in three types: high-mannose, complex andhybrid. All three types have a □1,4-N,N′-diacetylchitobiose (GlcNAc₂)core, connected to a mannose trisaccharide (Man₃). The core GlcNAc mayoptionally be fucosylated, but this is not always the case. High-mannoseglycans contain at least 2 further mannose residues, typically resultingin 5 to 9 mannose residues. Complex glycans have one or more sugarmonomers, not being mannose, connected to two of the mannose residues ofthe central Man₃ unit. These further sugar monomers are typicallyselected from GlcNAc, galactose (Gal), and sialic acid (Neu5Ac). Complexglycans exist in bi-, tri- and tetraantennary forms, depending on thenumber of (oligo)saccharide(s) that are connected to the central Man₃unit. Hybrid glycans have high-mannose type oligosaccharide connected toone of the mannose residue of the central Man₃ unit, and a complex typeoligosaccharide connected to the other mannose residue. An overview ofthe glycan types is given in FIG. 1. Even within a specific glycan type,many possibilities exist, increasing the heterogenicity ofglycoproteins. For example, biantennary complex glycans attached to theN297 residue of antibodies may exists in several distinct glycoforms,including but not limited to G1, G1F, G0F and SG1F, as depicted in FIG.2. Endo-β-N-acetylglucosaminidases (ENGases, also referred toendoglycosidases or Endos), typically hydrolyse the N-glycan of aglycoprotein at the β-1,4-glycosidic bond of the core chitobiose. Theseenzymes, found mainly in GH18 and GH85 in the CAZy classification(Lombard et al. in Nucleic Acids Res. 2014, 42, D490, incorporated byreference) are widely distributed from bacteria to animals and areinvolved in various biological functions such as glycan metabolism orbacterial pathogenesis (Karamanos, Adv. Biochem. 2013, 1, 81).Endoglycosidases are typically specific for hydrolysis of one or twoglycan types. Some are specific for hydrolysis of high-mannose glycans(e.g. EndoA, EndoD, EndoT), while others in addition to high-mannosealso cleave hybrid glycans (e.g. EndoF1, EndoH). Endoglycosidasesspecific for hydrolysis of complex glycans also exist in severalvariants. An overview of different activities is disclosed in Freeze etal. in Curr. Protoc. Mol. Biol., 2010, 89:17.13A.1-17, incorporated byreference herein. EndoS cleaves biantennary, triantennary andtetraantennary complex glycans, but its activity is nearly exclusivelylimited to antibodies, in particular the heavy chain of IgG.

EndoX and EndoY are two distinct endoglycosidases, which are preferablyindividually selected from the group consisting of EndoA, EndoBi,EndoBH, EndoBT, EndoCE, EndoD, EndoE, EfEndo18A, EndoF1, EndoF2, EndoF3,EndoH, EndoLL, EndoM, EndoOm, EndoS, and EndoT. These endoglycosidasesand their amino acid sequences are known to the skilled person. Herebelow, some preferred amino acid sequences for specific endoglycosidasesare given.

In a preferred embodiment, EndoS has at least 80%, preferably at least90%, more preferably at least 95% sequence identity with SEQ ID No. 4 orSEQ ID No. 5, most preferably EndoS has 100% sequence identity with SEQID No. 4 or SEQ ID No. 5. In one embodiment, EndoS has SEQ ID No. 4 orSEQ ID No. 5. Preferably, EndoS has the indicated sequence identitieswith SEQ ID No. 4.

SEQ ID No. 4: MPSIDSLHYLSENSKKEFKEELSKAGQESQKVKEILAKAQQADKQAQELAKMKIPEKIPMKPLHGPLYGGYFRTWHDKTSDPTEKDKVNSMGELPKEVDLAFIFHDWTKDYSLFWKELATKHVPKLNKQGTRVIRTIPWRFLAGGDNSGIAEDTSKYPNTPEGNKALAKAIVDEYVYKYNLDGLDVDVEHDSIPKVDKKEDTAGVERSIQVFEEIGKLIGPKGVDKSRLFIMDSTYMADKNPLIERGAPYINLLLVQVYGSQGEKGGWEPVSNRPEKTMEERWQGYSKYIRPEQYMIGFSFYEENAQEGNLWYDINSRKDEDKANGINTDITGTRAERYARWQPKTGGVKGGIFSYAIDRDGVAHQPKKYAKQKEFKDATDNIFHSDYSVSKALKTVMLKDKSYDLIDEKDFPDKALREAVMAQVGTRKGDLERFNGTLRLDNPAIQSLEGLNKFKKLAQLDLIGLSRITKLDRSVLPANMKPGKDTLETVLETYKKDNKEEPATIPPVSLKVSGLTGLKELDLSGFDRETLAGLDAATLTSLEKVDISGNKLDLAPGTENRQIFDTMLSTISNHVGSNEQTVKFDKQKPTGHYPDTYGKTSLRLPVANEKVDLQSQLLFGTVTNQGTLINSEADYKAYQNHKIAGRSFVDSNYHYNNFKVSYENYTVKVTDSTLGTTTDKTLATDKEETYKVDFFSPADKTKAVHTAKVIVGDEKTMMVNLAEGATVIGGSADPVNARKVFDGQLGSETDNISLGWDSKQSIIFKLKEDGLIKHWRFFNDSARNPETTNKPIQEASLQIFNIKDYNLDNLLENPNKFDDEKYWITVDTYSAQGERATAFSNTLNNITSKYWRVVFDTKGDRYSSPVVPELQILGYPLPNADTIMKTVTTAKELSQQKDKFSQKMLDELKIKEMALETSLNSKIFDVTAINANAGVLKDCIEKRQLLKK SEQ ID No. 5:MGSSHHHHHHSSGLVPRGSHMPSIDSLHYLSENSKKEFKEELSKAGQESQKVKEILAKAQQADKQAQELAKMKIPEKIPMKPLHGPLYGGYFRTWHDKTSDPTEKDKVNSMGELPKEVDLAFIFHDWTKDYSLFWKELATKHVPKLNKQGTRVIRTIPWRFLAGGDNSGIAEDTSKYPNTPEGNKALAKAIVDEYVYKYNLDGLDVDVEHDSIPKVDKKEDTAGVERSIQVFEEIGKLIGPKGVDKSRLFIMDSTYMADKNPLIERGAPYINLLLVQVYGSQGEKGGWEPVSNRPEKTMEERWQGYSKYIRPEQYMIGFSFYEENAQEGNLWYDINSRKDEDKANGINTDITGTRAERYARWQPKTGGVKGGIFSYAIDRDGVAHQPKKYAKQKEFKDATDNIFHSDYSVSKALKTVMLKDKSYDLIDEKDFPDKALREAVMAQVGTRKGDLERFNGTLRLDNPAIQSLEGLNKFKKLAQLDLIGLSRITKLDRSVLPANMKPGKDTLETVLETYKKDNKEEPATIPPVSLKVSGLTGLKELDLSGFDRETLAGLDAATLTSLEKVDISGNKLDLAPGTENRQIFDTMLSTISNHVGSNEQTVKFDKQKPTGHYPDTYGKTSLRLPVANEKVDLQSQLLFGTVTNQGTLINSEADYKAYQNHKIAGRSFVDSNYHYNNFKVSYENYTVKVTDSTLGTTTDKTLATDKEETYKVDFFSPADKTKAVHTAKVIVGDEKTMMVNLAEGATVIGGSADPVNARKVFDGQLGSETDNISLGWDSKQSIIFKLKEDGLIKHWRFFNDSARNPETTNKPIQEASLQIFNIKDYNLDNLLENPNKFDDEKYWITVDTYSAQGERATAFSNTLNNITSKYWRVVFDTKGDRYSSPVVPELQILGYPLPNADTIMKTVTTAKELSQQKDKFSQKMLDELKIKEMALETSLNSKIFDVTAI NANAGVLKDCIEKRQLLKK

In a preferred embodiment, EndoH has at least 80%, preferably at least90%, more preferably at least 95% sequence identity with SEQ ID No. 6,most preferably EndoH has 100% sequence identity with SEQ ID No. 6. Inone embodiment, EndoH has SEQ ID No. 6. SEQ ID No. 6:

APAPVKQGPTSVAYVEVNNNSMLNVGKYTLADGGGNAFDVAVIFAANINYDTGTKTAYLHFNENVQRVLDNAVTQIRPLQQQGIKVLLSVLGNHQGAGFANFPSQQAASAFAKQLSDAVAKYGLDGVDFDDEYAEYGNNGTAQPNDSSFVHLVTALRANMPDKIISLYNIGPAASRLSYGGVDVSDKFDYAWNPYYGTWQVPGIALPKAQLSPAAVEIGRTSRSTVADLARRTVDEGYGVYLTYNLDGGD RTADVSAFTRELYGSEAVRTP

In a preferred embodiment, EndoF1 has at least 80%, preferably at least90%, more preferably at least 95% sequence identity with SEQ ID No. 7,most preferably EndoF1 has 100% sequence identity with SEQ ID No. 7. Inone embodiment. EndoF1 has SEQ ID No. 7. SEQ ID No. 7:

AVTGTTKANIKLFSFTEVNDTNPLNNLNFTLKNSGKPLVDMVVLFSANINYDAANDKVFVSNNPNVQHLLTNRAKYLKPLQDKGIKVILSILGNHDRSGIANLSTARAKAFAQELKNTCDLYNLDGVFFDDEYSAYQTPPPSGFVTPSNNAAARLAYETKQAMPNKLVTVYVYSRTSSFPTAVDGVNAGSYVDYAIHDYGGSYDLATNYPGLAKSGMVMSSQEFNQGRYATAQALRNIVTKGYGGHMIFAMDPNRSNFTSGQLPALKLIAKELYGDELVYSNTPYSKDW

In a preferred embodiment, EndoF2 has at least 80%, preferably at least90%, more preferably at least 95% sequence identity with SEQ ID No. 8,most preferably EndoF2 has 100% sequence identity with SEQ ID No. 8. Inone embodiment, EndoF2 has SEQ ID No. 8. SEQ ID No. 8:

MAVNLSNLIAYKNSDHQISAGYYRTWRDSATASGNLPSMRWLPDSLDMVMVFPDYTPPENAYWNTLKTNYVPYLHKRGTKVIITLGDLNSATTTGGQDSIGYSSWAKGIYDKWVGEYNLDGIDIDIESSPSGATLTKFVAATKALSKYFGPKSGTGKTFVYDTNQNPTNFFIQTAPRYNYVFLQAYGRSTTNLTTVSGLYAPYISMKQFLPGFSFYEENGYPGNYWNDVRYPQNGTGRAYDYARWQPATGKKGGVFSYAIERDAPLTSSNDNTLRAPNFRVTKDLIKIMNP

In a preferred embodiment, EndoF3 has at least 80%, preferably at least90%, more preferably at least 95% sequence identity with SEQ ID No. 9,most preferably EndoF3 has 100% sequence identity with SEQ ID No. 9. Inone embodiment, EndoF3 has SEQ ID No. 9. SEQ ID No. 9:

MATALAGSNGVCIAYYITDGRNPTFKLKDIPDKVDMVILFGLKYWSLQDTTKLPGGTGMMGSFKSYKDLDTQIRSLQSRGIKVLQNIDDDVSWQSSKPGGFASAAAYGDAIKSIVIDKWKLDGISLDIEHSGAKPNPIPTFPGYAATGYNGWYSGSMAATPAFLNVISELTKYFGTTAPNNKQLQIASGIDVYAWNKIMENFRNNFNYIQLQSYGANVSRTQLMMNYATGTNKIPASKMVFGAYAEGGTNQANDVEVAKWTPTQGAKGGMMIYTYNSNVSYANAVRDAVKN

In a preferred embodiment, EfEndo18A has at least 80%, preferably atleast 90%, more preferably at least 95% sequence identity with SEQ IDNo. 10, most preferably EfEndo18A has 100% sequence identity with SEQ IDNo. 10. In one embodiment, EfEndo18A has SEQ ID No. 10. SEQ ID No. 10:

ASTVTPKTVMYVEVNNHDFNNVGKYTLAGTNQPAFDMGIIFAANINYDTVNKKPYLYLNERVQQTLNEAETQIRPVQARGTKVLLSILGNHEGAGFANFPTYESADAFAAQLEQVVNTYHLDGIDFDDEYAEYGKNGTPQPNNSSFIWLLQALRNRLGNDKLITFYNIGPAAANSSANPQMSSLIDYAWNPYYSTWNPPQIAGMPASRLGASAVEVGVNQNLAAQYAKRTKAEQYGIYLMYNLPGKDSSAYISAATQELYGRKTNYSPTVPTP

These preferred sequences for the individual endoglycosidases also applyto the fusion enzyme according to the invention. Thus, for example incase EndoX is EndoS, it is preferred that the amino acid sequence ofEndoS is as defined here above. The skilled person is capable ofapplying the sequences provided above to the fusion enzyme according toformula (1).

In one embodiment, the enzyme according to the invention comprises anamino acid sequence selected from SEQ ID NO:4-SEQ ID NO:10, connectedvia an amino acid sequence selected from SEQ ID NO:11 and SEQ ID NO:12to another amino acid sequence selected from SEQ ID NO:4-SEQ ID NO:10,individually having at least 50% sequence identity, preferably at least70%, more preferably at least 80% sequence identity with the individualSEQ IDs, such as at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identitywith respect to each one of SEQ ID NO:4-SEQ ID NO:12. In a preferredembodiment, these sequence identities apply to the combination of SEQIDs in the fusion enzyme according to the invention.

Preferably, the enzyme of the invention, having the above indicatedsequence identities with respect to SEQ ID NO: 2, has EndoS and EndoHactivity. Most preferably, the enzyme according to the invention has100% sequence identity with SEQ ID NO: 2. In one embodiment, the enzymeaccording to the invention comprising SEQ ID NO:4 connected via SEQ IDNO:11 to SEQ ID NO:6, individually having at least 50% sequenceidentity, preferably at least 70%, more preferably at least 80% sequenceidentity with the individual SEQ IDs, such as at least 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% sequence identity with respect to SEQ ID NO:4, SEQ ID NO:11and SEQ ID NO:6. In a preferred embodiment, these sequence identitiesapply to the combination of SEQ ID NO:4, SEQ ID NO:11 and SEQ ID NO:6.Preferably, the enzyme of the invention, having the above indicatedsequence identities with respect to SEQ ID NO: 1, has EndoS and EndoHactivity. Most preferably, the enzyme according to the invention has100% sequence identity with SEQ ID NO:1. In one embodiment, the enzymeaccording to the invention comprising SEQ ID NO:4 connected via SEQ IDNO:12 to SEQ ID NO:6, individually having at least 50% sequenceidentity, preferably at least 70%, more preferably at least 80% sequenceidentity with the individual SEQ IDs, such as at least 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% sequence identity with respect to SEQ ID NO:4, SEQ ID NO:12and SEQ ID NO:6.

In a preferred embodiment, the fusion enzyme according to the inventionhas at least 50% sequence identity, preferably at least 70%, morepreferably at least 80% sequence identity, such as at least 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99%, or most preferably 100% sequence identity with respectto any one of SEQ ID NO:1, 2 and 13-21. Preferred sequence IDs areselected from SEQ ID NO:1, 2, 17, 19 and 21. Most preferred sequence IDsare selected from SEQ ID NO:1, 2 and 21.

In one embodiment, the fusion enzyme according to the invention has atleast 50% sequence identity, preferably at least 70%, more preferably atleast 80% sequence identity, such as at least 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%,or most preferably 100% sequence identity with respect to SEQ ID NO:1.In one embodiment, the fusion enzyme according to the invention has atleast 50% sequence identity, preferably at least 70%, more preferably atleast 80% sequence identity, such as at least 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%,or most preferably 100% sequence identity with respect to SEQ ID NO:2.In one embodiment, the fusion enzyme according to the invention has atleast 50% sequence identity, preferably at least 70%, more preferably atleast 80% sequence identity, such as at least 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%,or most preferably 100% sequence identity with respect to SEQ ID NO:13.In one embodiment, the fusion enzyme according to the invention has atleast 50% sequence identity, preferably at least 70%, more preferably atleast 80% sequence identity, such as at least 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%,or most preferably 100% sequence identity with respect to SEQ ID NO:14.In one embodiment, the fusion enzyme according to the invention has atleast 50% sequence identity, preferably at least 70%, more preferably atleast 80% sequence identity, such as at least 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%,or most preferably 100% sequence identity with respect to SEQ ID NO:15.In one embodiment, the fusion enzyme according to the invention has atleast 50% sequence identity, preferably at least 70%, more preferably atleast 80% sequence identity, such as at least 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%,or most preferably 100% sequence identity with respect to SEQ ID NO:16.In one embodiment, the fusion enzyme according to the invention has atleast 50% sequence identity, preferably at least 70%, more preferably atleast 80% sequence identity, such as at least 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%,or most preferably 100% sequence identity with respect to SEQ ID NO:17.In one embodiment, the fusion enzyme according to the invention has atleast 50% sequence identity, preferably at least 70%, more preferably atleast 80% sequence identity, such as at least 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%,or most preferably 100% sequence identity with respect to SEQ ID NO:18.In one embodiment, the fusion enzyme according to the invention has atleast 50% sequence identity, preferably at least 70%, more preferably atleast 80% sequence identity, such as at least 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%,or most preferably 100% sequence identity with respect to SEQ ID NO:19.In one embodiment, the fusion enzyme according to the invention has atleast 50% sequence identity, preferably at least 70%, more preferably atleast 80% sequence identity, such as at least 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%,or most preferably 100% sequence identity with respect to SEQ ID NO:20.In one embodiment, the fusion enzyme according to the invention has atleast 50% sequence identity, preferably at least 70%, more preferably atleast 80% sequence identity, such as at least 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%,or most preferably 100% sequence identity with respect to SEQ ID NO:21.

In one embodiment, EndoX and EndoY are two distinct endoglycosidases andboth are selected from the group consisting of EndoA, EndoBi, EndoBH,EndoBT, EndoCE, EndoD, EndoE, EfEndo18A, EndoF1, EndoF2, EndoF3, EndoH,EndoLL, EndoM, EndoOm, EndoS, and EndoT. A preferred group ofendoglycosidases to be used as EndoX and EndoY consists of EndoE,EfEndo18A, EndoF1, EndoF2, EndoF3, EndoH, EndoS and EndoT, morepreferably of EndoF1, EndoF2, EndoF3, EfEndo18A, EndoH and EndoS. In oneembodiment, at least one of EndoX and EndoY, preferably EndoX, isselected from the group consisting of EndoF2, EndoF3 and EndoS. In oneembodiment, at least one of EndoX and EndoY, preferably EndoY, isselected from the group consisting of EfEndo18A EndoF1 and EndoH.

In one embodiment, one of EndoX and EndoY is an endoglycosidase capableof cleaving a glycan of the high-mannose type, such as EndoA, EndoE,EfEndo18A, EndoF1, EndoH, EndoM, or EndoT.

Preferably, the endoglycosidase capable of cleaving a glycan of thehigh-mannose type is selected from the group consisting of EndoE,EfEndo18A, EndoF1, EndoH and EndoT, more preferably selected from thegroup consisting of EfEndo18A, EndoF1 and EndoH. Most preferably, theendoglycosidase capable of cleaving a glycan of the high-mannose type isEndoH. Preferably, the other of EndoX and EndoY is an endoglycosidasehaving a different activity, preferably an endoglycosidase capable ofcleaving a glycan of the complex type.

In one embodiment, one of EndoX and EndoY is an endoglycosidase capableof cleaving a glycan of the complex type, such as EndoE, EndoF2, EndoF3and EndoS. Preferably, the endoglycosidase capable of cleaving a glycanof the complex type is selected from the group consisting of EndoF2,EndoF3, and EndoS, more preferably selected from the group consisting ofEndoF3 and EndoS. Most preferably, the endoglycosidase capable ofcleaving a glycan of the complex type is EndoS. Preferably, the other ofEndoX and EndoY is an endoglycosidase having a different activity,preferably an endoglycosidase capable of cleaving a glycan of thehigh-mannose type.

It is especially preferred that the fusion enzyme according to theinvention contains two distinct endoglycosidases which differ inendoglycosidase activity, as two distinct endoglycosidase activities canas such be combined in a single enzyme. Thus, EndoX and EndoY preferablyeach have a distinct endoglycosidase activity selected from the capacityof hydrolysing high-mannose glycans, the capacity of hydrolysing complexglycans and the capacity of hydrolysing hybrid glycans, more preferablyselected from the capacity of hydrolysing high-mannose glycans and thecapacity of hydrolysing complex glycans. Preferably, one of EndoX andEndoY is an endoglycosidase that capable of hydrolysing high-mannoseglycans, and the other endoglycosidase is capable of hydrolysing complexglycans. Preferably, the endoglycosidase that is capable of hydrolysinghigh-mannose glycans is also capable of hydrolysing hybrid glycans.Preferably, the endoglycosidase that is capable of hydrolysing complexglycans is capable of hydrolysing biantennary and/or triantennarycomplex glycans, most preferably all complex glycans.

For example, when EndoX is EndoS and EndoY is EndoH, the resultingfusion enzyme exhibits both EndoS and EndoH activity, and is capable oftrimming complex glycans on glycoproteins (such as antibodies) at thecore GlcNAc unit, leaving only the core GlcNAc residue on theglycoprotein (EndoS activity) as well as well as trimming (splittingoff) high-mannose glycans (EndoH activity). Surprisingly, bothactivities of the fusion enzyme function smoothly at a pH around 7-8,while monomeric EndoH requires a pH in the range of 5-6, or even a pH of6 to operate optimally. In one embodiment, EndoX and EndoY are twodistinct endoglycosidases that differ in optimal pH of at least 1 pHunits, preferably at least 1.5 pH unit, most preferably at least 2 pHunits. The skilled person is aware of the pH optimum that belongs tospecific endoglycosidases. Such fusion enzymes may be active at aspecific pH, which is not the optimal pH of at least one of EndoX andEndoY.

In a preferred embodiment, one of EndoX and EndoY is selected fromEndoF2, EndoF3 or EndoS, and the other of EndoX and EndoY is selectedfrom EndoD, EndoH, EndoE, EfEndo18A, EndoT or EndoF1. Preferably, EndoXis selected from EndoF2, EndoF3 or EndoS, and EndoY is selected fromEndoD, EndoH, EndoE, EfEndo18A, EndoT or EndoF1. A such, the fusionenzyme is capable of hydrolysing complex glycans (EndoF2, EndoF3 andEndoS activity) as well as hydrolysing high-mannose glycans (EndoD,EndoF1, EndoH, EndoE, EfEndo18A, EndoT or EndoF1 activity). In oneembodiment, EndoX is EndoS, and EndoY is preferably EndoD, EndoF1,EndoH, EndoE, EfEndo18A, EndoT or EndoF1, more preferably EndoY isEndoF1, EndoH or EfEndo18A, most preferably EndoY is EndoH. Mostpreferably, EndoX is EndoS and EndoY is EndoH. Alternatively, EndoX isEndoF2 and EndoY is preferably EndoD, EndoH, EndoE, EfEndo18A, EndoT orEndoF1, more preferable EndoY is EndoF1, EndoH or EfEndo18A, mostpreferably EndoY is EndoF1. Most preferably, EndoX is EndoF2 and EndoYis EndoF1. Alternatively, EndoX is EndoF3 and EndoY is preferably EndoD,EndoH, EndoE, EfEndo18A, EndoT or EndoF1, more preferably EndoY isEndoF1, EndoH or EfEndo18A, most preferably EndoY is EndoH. Mostpreferably, EndoX is EndoF3 and EndoY is EndoH.

In one embodiment, Endo X and EndoY are both individually selected fromEndoF1, EndoF2, EndoF3, EfEndo18A, EndoS and EndoH. Preferably, EndoX isselected from EndoF2, EndoF3 and EndoS and EndoY is selected fromEndoF1, EfEndo18A and EndoH.

In one embodiment, one of EndoX and EndoY is EndoS or EndoF3, and theother one of EndoX and EndoY is EndoF1 or EndoH. Preferably, EndoX isEndoS or EndoF3, and EndoY is EndoF1 or EndoH.

In a preferred embodiment, the fusion enzyme according to the inventionis selected from the group consisting of enzymes of structure (1),wherein EndoX=EndoF3 and EndoY=EndoH; EndoX=EndoF3 and EndoY=EndoE;EndoX=EndoF3 and EndoY=EfEndo18A; EndoX=EndoF3 and EndoY=EndoT;EndoX=EndoF3 and EndoY=EndoF1; EndoX=EndoS and EndoY=EndoH; EndoX=EndoSand EndoY=EndoE; EndoX=EndoS and EndoY=EfEndo18A; EndoX=EndoS andEndoY=EndoT; EndoX=EndoS and EndoY=EndoF1; EndoX=EndoF2 and EndoY=EndoH;EndoX=EndoF2 and EndoY=EndoE; EndoX=EndoF2 and EndoY=EfEndo18A;EndoX=EndoF2 and EndoY=EndoT; and EndoX=EndoF2 and EndoY=EndoF1. Morepreferably, the fusion enzymes according to the invention is selectedfrom the group consisting of enzymes of structure (1), whereinEndoX=EndoF3 and EndoY=EndoH; EndoX=EndoF3 and EndoY=EfEndo18A;EndoX=EndoF3 and EndoY=EndoF1; EndoX=EndoS and EndoY=EndoH; EndoX=EndoSand EndoY=EfEndo18A; EndoX=EndoS and EndoY=EndoF1; EndoX=EndoF2 andEndoY=EndoH; EndoX=EndoF2 and EndoY=EfEndo18A; and EndoX=EndoF2 andEndoY=EndoF1. Even more preferably, the fusion enzymes according to theinvention is selected from the group consisting of enzymes of structure(1), wherein EndoX=EndoF3 and EndoY=EndoH; EndoX=EndoS and EndoY=EndoH;EndoX=EndoS and EndoY=EfEndo18A; EndoX=EndoS and EndoY=EndoF1; andEndoX=EndoF2 and EndoY=EndoF1. Most preferably, the fusion enzymesaccording to the invention is an enzyme of structure (1), whereinEndoX=EndoS and EndoY=EndoH.

Linker

In the enzyme according to the invention, EndoX and EndoY are preferablylinked by a linker. In case a linker is present, p=1. In case no linkeris present, p=0. Preferably, p=1. Linkers for fusion enzymes are knownin the art, and any suitable linker may be used, including flexible andrigid linkers. Further guidance can be found in Chen et al., Adv. DrugDeliv. Rev. 2013, 65, 1357-1369 and Fusion Protein Technologies forBiopharmaceuticals: Application and Challenges, Chapter 4: FusionProtein Linkers: Effects on Production, Bioactivity, andPharmacokinetics, 2013, John Wiley & Sons, Inc, both of which areincorporated herein in their entirety. Preferably, said linker is aflexible linker allowing the adjacent protein to move relative freely.

In one embodiment, the linker, preferably the flexible linker, iscomposed of amino residues and has a length of 1 to 100 amino acidresidues, preferably 3 to 59, 10 to 45 or 15 to 40 amino acid residues,such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acid residues.

In one embodiment, the linker, preferably the flexible linker, iscomposed of amino residues like glycine, serine, histidine and/oralanine and has a length of 3 to 59 amino acid residues, preferably 10to 45 or 15 to 40 amino acid residues, such as 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39 or 40 amino acid residues.

The linker preferably comprises one or more flexible domains, thatprovide flexibility to the linker. Preferably, one or two, mostpreferably two of such flexible domains are comprised in the linker.Such flexible domains are known in the art and are typically composed ofglycine, serine and/or threonine. In one embodiment, the linkercomprises at least one glycine, serine and/or threonine residue.Preferably, at least 40% of the amino acids of the linker are selectedfrom glycine, serine and threonine, more preferably 50-90%, mostpreferably 70-85% of the amino acids of the linker are selected fromglycine, serine and threonine. In one embodiment, the linker does notcomprise threonine and the above ranges apply to glycine and serine.

Specific suitable flexible domains include GS-domains (such as (G4S)_(n)wherein n is an integer in the range 1-10, preferably 1-6, mostpreferably 2-4), poly-G (such as Gm, wherein m is an integer in therange 1-30, preferably 3-20, most preferably 5-10), GSAGSAAGSGEF,EGKSSGSGSESKST, PAS linkers (Pro, Ala, Ser based linkers; see Schlapschyet al., Protein Eng Des Sel. 2013, 26, 489-501, incorporated byreference) and extended recombinant polypeptide (XTEN) linkers (seePodust et al., Protein Eng Des Sel. 2013, 26, 743-753, incorporated byreference). GS-domains, consisting of stretches of glycine and serineresidues, are most preferred. So, in one embodiment, the linkercomprises one or more (G4S)_(n) domains, preferably one or two, mostpreferably two domains.

Alternatively or additionally, the linker may comprise one or more rigiddomains, such as □-helix forming domains, such as (EAAAK)_(o) orA(EAAAK)_(o)A (wherein o is an integer in the range 1-10, preferably2-5, most preferably 3 or 4), and proline-rich domains, such as (XP)_(q)(wherein X is any amino acid, preferably selected from alanine, lysineand glutamine, and q is an integer in the range 2-25, preferably 5-17).

Optionally, the linker comprises a tag for ease of purification and/ordetection as known in the art, such as an Fc-tag, FLAG-tag,poly(His)-tag, (RP)₆R-tag, HA-tag and Myc-tag. Such a tag may also bepresent elsewhere in the linker according to the invention. Thus, in oneembodiment, the fusion enzyme according to the invention comprises a tagfor ease of purification and/or detection, such as an Fc-tag, FLAG-tag,poly(His)-tag, (RP)₆R-tag, HA-tag and Myc-tag, most preferably apoly(His)-tag. In one embodiment, the fusion enzyme according to theinvention comprises a linker, i.e. p=1, and the linker comprises a tagfor ease of purification and/or detection, such as an Fc-tag, FLAG-tag,poly(His)-tag, (RP)₆R-tag, HA-tag and Myc-tag, most preferably apoly(His)-tag. The tag may be located at the C-terminus of the linker,at the N-terminus of the linker or may be embedded in the linker withfurther amino acid(s) at either side of the tag. The latter conformationis preferred, especially when flexible domains are located at eitherside of the tag, as it brings optimal accessibility of the tag forbinding to an affinity matrix.

In one embodiment, the linker has the structure(G₄S)_(n1)(H)_(r)(EF)_(s)(G₄S)_(n2), wherein n1 and n2 individually areintegers in the range 1-10, preferably 1-6, even more preferably 2-4,most preferably 3, and r is an integer in the range of 2-10, preferably4-8, most preferably 6, and s=0 or 1. In one embodiment, the linker hasthe structure (G₄S)₃(H)₆(G₄S)₃, i.e. wherein n1=3, n2=3, r=6 and s=0(amino acids 950 to 985 of SEQ ID No. 2). In one embodiment, the linkerhas the structure (G₄S)₃(H)₆EF(G₄S)₃, i.e. wherein n1=3, n2=3, r=6 ands=1 (amino acids 950 to 987 of SEQ ID No. 1).

In a preferred embodiment, the linker has at least 80%, preferably atleast 90%, more preferably at least 95% sequence identity with SEQ IDNo. 11 or 12, most preferably the linker has 100% sequence identity withSEQ ID No. 11 or SEQ ID No. 12. In one embodiment, the linker has SEQ IDNo. 11. In one embodiment, the linker has SEQ ID No. 12.

SEQ ID No.11: GGGGSGGGGSGGGGSHHHHHHEFGGGGSGGGGSGGGGS SEQ ID No.12:GGGGSGGGGSGGGGSHHHHHHGGGGSGGGGSGGGGS

The fusion enzyme according to the invention can be prepared by routinetechniques known in the art, such as introducing an expression vector(e.g. plasmid) comprising the enzyme coding sequence into a host cell(e.g. E. coli) for expression, from which the enzyme can be isolated.Alternatively, the enzyme is produced by transient expression in CHO. Apossible approach for the preparation and purification of the fusionenzyme according to the invention is given in examples 1-4 and 16-24,and its functioning is demonstrated in examples 5, 6, 8, 13-15 and25-37, wherein various glycoproteins, including trastuzumab andhigh-mannose trastuzumab, are efficiently trimmed in a single step.

Preferred Fusion Enzyme

In an especially preferred embodiment, the invention concerns a fusionenzyme comprising the two endoglycosidases EndoS and EndoH. In aparticular example the two endoglycosidases EndoS and EndoH areconnected via a linker, preferably a-(Gly₄Ser)₃-(His)₆-(Gly₄Ser)₃-linker. The fusion enzyme according to theinvention as also referred to as EndoSH. In one embodiment, the enzymeaccording to the invention has at least 50% sequence identity with SEQID NO: 1, preferably at least 70%, more preferably at least 80% sequenceidentity with SEQ ID NO: 1, such as at least 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity with SEQ ID NO: 1. Preferably, the enzyme of theinvention, having the above indicated sequence identity to SEQ ID NO: 1,has EndoS and EndoH activity. Most preferably, the enzyme according tothe invention has 100% sequence identity with SEQ ID NO: 1. In oneembodiment, the enzyme according to the invention has at least 50%sequence identity with SEQ ID NO: 2, preferably at least 70%, morepreferably at least 80% sequence identity with SEQ ID NO: 2, such as atleast 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 2.Preferably, the enzyme of the invention, having the above indicatedsequence identity to SEQ ID NO: 2, has EndoS and EndoH activity. Mostpreferably, the enzyme according to the invention has 100% sequenceidentity with SEQ ID NO: 2.

Also encompassed are fusion enzymes of EndoS and EndoH, wherein thelinker is replaced by another suitable linker known in the art, whereinsaid linker may be rigid or flexible. Preferably, said linker is aflexible linker allowing the adjacent protein domains to move relativefreely to one another. Preferably, said flexible linker is composed ofamino residues like glycine, serine, histidine and/or alanine and has alength of 3 to 59 amino acid residues, preferably 10 to 45 or 15 to 40amino acid residues, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acidresidues, or 20 to 38, 25 to 37 or 30 to 36 amino acid residues.Optionally, the fusion enzyme is covalently linked to, or comprises, atag for ease of purification and or detection as known in the art, suchas an Fc-tag, FLAG-tag, poly(His)-tag, HA-tag and Myc-tag. Trimming ofglycoproteins is known in the art, from e.g. Yamamoto, Biotechnol. Lett.2013, 35, 1733, WO 2007/133855 or WO 2014/065661, which are incorporatedherein in their entirety. The enzyme according to this embodimentexhibits both EndoS and EndoH activity, and is capable of trimmingglycans on glycoproteins (such as antibodies) at the core GlcNAc unit,leaving only the core GlcNAc residue on the glycoprotein (EndoSactivity) as well as well as high-mannose glycans (EndoH activity).Surprisingly, both activities of the fusion enzyme function smoothly ata pH around 7-8, while monomeric EndoH requires a pH of 6 to operateoptimally.

Use

A further aspect of the invention concerns the use of the fusion enzymeaccording to the invention for trimming glycoproteins, preferably fortrimming antibodies. Trimming may also be referred to as deglycosylationand is further defined here below, in the context of the processaccording to the invention. The use according to this aspect may occurin vitro or in vivo.

Process for Trimming of Glycoproteins

The fusion enzyme according to the invention is particularly suited fortrimming of glycoproteins. Thus, in a further aspect, the inventionconcerns a process for the trimming of glycoproteins. The processaccording to this aspect may occur in vitro or in vivo. Trimming ofglycoproteins is known in the art, from e.g. Yamamoto, Biotechnol. Lett.2013, 35, 1733, WO 2007/133855 or WO 2014/065661, which are incorporatedherein in their entirety. Glycoproteins, such as antibodies, typicallycontain different glycoforms, which require different endoglycosidasesto remove. The fusion enzymes of the invention are especially suitableto deglycosylate in a single step a glycoprotein having two differentglycan chains. Thus, in one embodiment, the glycoprotein that issubjected to the process according to the invention comprises at leasttwo distinct glycans, preferably two distinct glycans. Preferably, theglycoprotein comprises at least one high-mannose glycan and at least onecomplex glycan, more preferably the glycoprotein comprises at least onehigh-mannose glycan, at least one hybrid glycan and at least one complexglycan. The complex glycan may be a bi-, tri-, or tetraantennary glycan.

In an especially preferred embodiment, the glycoprotein is an antibody.

The process according to the present aspect may also be referred to as aprocess for modifying a glycoprotein. The process comprised contactingthe glycoprotein with a fusion enzyme according to the invention, toobtain a trimmed glycoprotein. The process may also be referred to as aprocess for trimming a glycoprotein or deglycosylation of aglycoprotein. Trimming or deglycosylation of a glycoprotein refers tothe removal of a glycan from said glycoprotein. The exact structure ofthe glycan that is removed may vary depending on the exact nature of theendoglycosidases that are present in the fusion enzyme, but the coreGlcNAc residue is retained on the glycoprotein at all times. The skilledperson will appreciate which fusion enzyme, i.e. which combination ofendoglycosidases, is suitable for trimming of which glycosylationpattern of the glycoprotein.

With conventional endoglycosidases, glycoproteins containing acombination of a high-mannose glycan and a complex bi-, tri- ortetraantennary glycan would require two distinct enzymes for trimming,often requiring different buffer conditions and pH ranges. Theseglycoproteins can now efficiently be trimmed in a single step, withoutthe need to apply buffer exchange to achieve the optimal pH, with thefusion enzyme according to the invention. Thus, in one embodiment, theglycoprotein, preferably the antibody, comprises at least onehigh-mannose glycan and at least one complex bi-, tri- or tetraantennaryglycan, more preferably at least one high-mannose glycan, at least onehybrid and/or complex bi-, tri- or tetraantennary glycan. For example,the fusion enzyme wherein one of EndoX and EndoY is selected fromEndoF2, EndoF3 or EndoS, and the other of EndoX and EndoY is selectedfrom EndoH, EndoE, EfEndo18A, EndoT or EndoF1, is suitable for trimminga glycoprotein comprising a complex N-linked complex glycan and ahigh-mannose glycan, to obtain a trimmed glycoprotein comprising onlythe optionally fucosylated core N-acetylglucosamine substituent(s).

The skilled person is aware of suitable conditions to perform thetrimming of glycoproteins. For example, the process is carried out in amedium and at a temperature that is effective for trimmingglycoproteins. Typically, the media and conditions that apply for one ofthe individual endoglycosidase enzymes are applicable. As the optimal pHof the individual endoglycosidases may differ, the process may in oneembodiment be carried out at a pH which is 0.5-3 pH units, preferably1-2 pH units, different from the optimal pH of one or both, preferablyone of EndoX and EndoY. For example, in case one of EndoX and EndoY isEndoH, which has an optimal pH or 5-6, the process may be carried out atpH 7-8. In one embodiment, the trimming performed at a pH in the rangeof 4-9, preferably in the range of 6-8, most preferably in the range of7-8. The inventors surprisingly found that the fusion enzyme accordingto the invention wherein EndoX=EndoS and EndoY=EndoH is fullyoperational at a pH above 7, whereas the functional pH range for EndoHis 5.0 to 6.0, with the optimum pH at 5.5.

Moreover, the inventors found that the activity of a particularendoglycosidase in a fusion protein can display a higher trimmingefficiency compared to the same endoglycosidase as a single enzyme.

The trimming affords trimmed glycoproteins, wherein all glycan moietiespresent in the original glycoprotein, irrespective of their type andglycoform, are trimmed and only the optionally fucosylated coreN-acetylglucosamine substituent(s) remain. Said optionally fucosylatedcore N-acetylglucosamine substituent is typically bonded via anN-glycosidic bond to the amide nitrogen atom in the side chain of anasparagine amino acid of the glycoprotein, such as N297 when theglycoprotein is an antibody.

The thus obtained trimmed glycoprotein can be used as deemed fit. Forexample, when the glycoprotein is the product of interest, the trimmedglycoprotein according to the invention is homogeneous with respect toglycosylation patterns. This can be particularly important when theglycoprotein is used as medicament, since the therapeutic efficacyand/or the toxicity may vary for different glycoforms of theglycoprotein. Such unpredictable variations in efficacy and toxicity areeradicated when the process according to the invention is utilized.

Alternatively, the trimmed glycoprotein can be used for furtherfunctionalization, such as by introduction of an optionally substitutedsugar moiety is known in the art, from e.g. van Geel et. al,Bioconjugate Chem, 2015, 26, 2233, incorporated by reference. Thetrimmed glycoprotein may be contacted with a compound of the formulaS—P, wherein S is an optionally substituted sugar moiety and P is anucleotide, in the presence of a suitable catalyst, such as aglycosyltransferase or N-acetylglycosyltransferase. The thus obtainedmodified glycoprotein comprises sugar moiety S connected to thenon-reducing end of the trimmed glycan. Using a substituted sugar moietyS, the possibilities for further modification or functionalization ofthe glycoprotein via said substituent are endless. Such a sequence ofreaction steps finds particular use in the preparation of bioconjugates,such as antibody-drug conjugates. Such steps are known to the skilledperson, e.g. from WO 2014/065661, incorporated by reference herein.

EXAMPLES

RP-HPLC Analysis of Reduced Monoclonal Antibodies:

Prior to RP-HPLC analysis samples were reduced by incubating a solutionof 10 μg (modified) IgG for 15 minutes at 37° C. with 10 mM DTT and 100mM Tris pH 8.0 in a total volume of 50 μL. A solution of 49% ACN, 49% MQand 2% formic acid (50 μL) was added to the reduced sample. Reversephase HPLC was performed on a Agilent 1100 HPLC using a ZORBAX Poroshell300SB-C8 1×75 mm, 5 μm (Agilent Technologies) column run at 1 ml/min at70° C. using a 16.9 minute linear gradient from 25 to 50% buffer B (withbuffer A=90% MQ, 10% ACN, 0.1% TFA and buffer B=90% ACN, 10% MQ, 0.1%TFA).

Mass Spectral Analysis of Monoclonal Antibodies:

Prior to mass spectral analysis, IgGs were either treated with DTT,which allows analysis of both light and heavy chain, or treated withFabricator™ (commercially available from Genovis, Lund, Sweden), whichallows analysis of the Fc/2 fragment. For analysis of both light andheavy chain, a solution of 20 μg (modified) IgG was incubated for 5minutes at 37° C. with 100 mM DTT in a total volume of 4 μL. If present,azide-functionalities are reduced to amines under these conditions. Foranalysis of the Fc/2 fragment, a solution of 20 μg (modified) IgG wasincubated for 1 hour at 37° C. with Fabricator™ (1.25 U/μL) inphosphate-buffered saline (PBS) pH 6.6 in a total volume of 10 μL. Afterreduction or Fabricator-digestion the samples were washed trice withmilliQ using an Amicon Ultra-0.5, Ultracel-10 Membrane (Millipore)resulting in a final sample volume of approximately 40 μL. Next, thesamples were analyzed by electrospray ionization time-of-flight(ESI-TOF) on a JEOL AccuTOF. Deconvoluted spectra were obtained usingMagtran software.

Example 1: Cloning of Fusion Protein EndoSH into (pET22B) ExpressionVector

A pET22B-vector containing an EndoS-(G₄S)₃-(His)₆-EF-(G₄S)₃-EndoH(EndoSH) coding sequence (EndoSH being identified by SEQ ID NO: 1)between EcoRI-HindIII sites was obtained from Genscript. The DNAsequence for the EndoSH fusion protein consists of the encoding residues48-995 of EndoS fused via an N-terminal linked glycine-serine (GS)linker to the coding residues 41-313 of EndoH. The glycine-serine (GS)linker comprises a -(G₄S)₃-(His)₆-EF-(G₄S)₃-format, allowing spacing ofthe two enzymes and at the same time introducing a IMAC-purificationtag.

Example 2: E. coli Expression of Fusion Protein EndoSH

Expression of the EndoSH fusion protein (identified by SEQ ID NO: 1)starts with the transformation of the plasmid (pET22b-EndoSH) into BL21cells. Next step is the inoculation of 500 mL culture (LBmedium+Ampilicin) with BL21 cells. When the OD600 reached 0.7 thecultures were induced with 1 mM IPTG (500 μL of 1M stock solution).

Example 3: Purification of Fusion Protein EndoSH from E. coli

After overnight induction at 16° C. the culture were pelleted bycentrifugation. The pellet was resuspended in 40 mL PBS and incubated onice with 5 ml lysozyme (10 mg/mL) for 30 minutes. After half an hour 5ml 10% Triton-X-100 was added and sonicated (10 minutes) on ice. Afterthe sonification the cell debris was removed by centrifugation (10minutes 8000×g) followed by filtration through a 0.22 μM-pore diameterfilter. Alternatively, lysis of the pellet containing EndoSH can beperformed by means of French press. Here the pellet is re-suspended in10 mL PBS/gram of pellet. The cell suspension is lysed three times underpressure (20000-25000 psi) by French press using Emulsiflex C3, Avestin.After French press the cell debris was removed by centrifugation (20minutes 10000×g). The soluble extract/fraction was loaded onto a HisTrapHP 5 mL column (GE Healthcare). The column was first washed with bufferA (20 mM Tris buffer, 20 mM imidazole, 500 mM NaCl, pH 7.5). Retainedprotein was eluted with buffer B (20 mM Tris, 500 mM NaCl, 250 mMimidazole, pH 7.5, 10 mL). Fractions were analyzed by SDS-PAGE onpolyacrylamide gels (12%). The fractions that contained purified targetprotein were combined and the buffer was exchanged against 20 mM Tris pH7.5 and 150 mM NaCl by dialysis performed overnight at 4° C. Thepurified protein was concentrated to at least 2 mg/mL using an AmiconUltra-0.5, Ultracel-10 Membrane (Millipore). The product is stored at−80° C. prior to further use.

Example 4: CHO Expression and Purification of Fusion Protein EndoSH fromCHO

EndoSH (identified by SEQ ID NO: 2) was transiently expressed in CHO K1cells by Evitria (Zurich, Switzerland) at 20 mL scale. The supernatant,containing fusion protein EndoSH, was diluted with elution buffer (2 mL,20 mM Tris, 500 mM NaCl, 500 mM imidazole) and binding buffer (18 mL, 20mM Tris, 500 mM NaCl, 5 mM imidazole, pH=7.4) to a final imidazoleconcentration of 10 mM. The mixture was loaded onto a Ni-NTA column (GEHealthcare) and the product was eluted following a standard elutionprotocol. The collected fractions (5 mL) were analysed on an SDS-PAGE(10%) gel. The faction containing product was partially concentrated (˜2mL) and dialyzed against TBS buffer. Protein concentration, determinedby nanodrop analysis, was set at 0.5 mg/mL.

Example 5: Trimming of Trastuzumab by EndoSH

Trastuzumab (obtained from Epirus biopharma (Utrecht, The Netherlands);14 mg/mL) in 25 mM Tris buffer pH 8, was trimmed using a concentrationof either 0.1 or 1 w/w % EndoSH. The reactions, 350 μg trastuzumab (25μL) and the appropriate amount of EndoSH, were stirred at 37° C. andanalyzed by MS analysis over time, 1 to 3 hours. Samples were subjectedto Fabricator treatment prior to analysis. Full conversions to thetrimmed product, which is trimmed to the core GlcNAc sugar residue, wasobserved after 1 hour at 37° C. with 0.1 w/w % EndoSH.

Example 6: Trimming of High-Mannose Trastuzumab by Fusion Protein EndoSH

Trastuzumab having high-mannose glycans (obtained via transientexpression in CHO K1 cells in the presence of kifunensine performed byEvitria (Zurich, Switzerland)) (14 mg/mL) in 25 mM Tris buffer pH 8, wastrimmed using a concentration of either 0.1 or 1 w/w % EndoSH. Thereactions, 350 μg high-mannose trastuzumab (25 μL) and the appropriateamount of EndoSH, were stirred at 37° C. and analyzed by MS analysisover time, 1-3 hours. Samples were subjected to Fabricator treatmentprior to analysis. Full conversions to the trimmed product, which istrimmed to the core GlcNAc sugar residue, was observed after 3 hours at37° C. with 1 w/w % EndoSH.

Example 7: Transient Expression and Purification of cAC10

cAC10 was transiently expressed in CHO K1 cells by Evitria (Zurich,Switzerland) at 5 L scale. The supernatant was purified using a XK 26/20column packed with 50 mL protein A sepharose. In a single run 5 Lsupernatant was loaded onto the column followed by washing with at least10 column volumes of 25 mM Tris pH 7.5, 150 mM NaCl. Retained proteinwas eluted with 0.1 M Glycine pH 2.7. The eluted cAC10 was immediatelyneutralized with 1.5 M Tris-HCl pH 8.8 and dialyzed against 25 mM TrispH 8.0. Next the IgG was concentrated to approximately 20 mg/mL using aVivaspin Turbo 15 ultrafiltration unit (Sartorius) and stored at −80° C.prior to further use.

Example 8: Trimming of cAC10 by EndoSH

Glycan trimming of cAC10 (obtained via transient expression in CHO K1cells performed by Evitria (Zurich, Switzerland)) was performed withfusion protein EndoSH. Thus, cAC10 (14.5 mg/mL) was incubated withEndoSH (1 w/w %) in 25 mM Tris pH 7.5 with 150 mM NaCl for approximately16 hours at 37° C. The trimmed IgG was dialyzed against 3×1 L of 25 mMTris-HCl pH 8.0. Mass spectral analysis of a fabricator-digested sampleshowed three peaks of the Fc/2-fragment belonging to one major product(observed mass 24105 Da, approximately 80% of total Fc/2 fragment),corresponding to core GlcNAc(Fuc)-substituted cAC10, and two minorproducts (observed masses of 23959 and 24233 Da, approximately 5 and 15%of total Fc/2 fragment), corresponding to core GlcNAc-substituted cAC10and core GlcNAc(Fuc)-substituted cAC10 with C-terminal lysine.

Examples 9-12: Preparation of cAC10 Bioconjugate

To demonstrate that the antibodies trimmed by the fusion enzymeaccording to the invention can be further modified,antibody-drug-conjugate 113 has been prepared from the trimmed antibodyof Example 8. Compound 99 was prepared via activation of compound 58 asdisclosed in and prepared according to Example 50 of WO 2016/053107(PCT/NL2015/050697). In the second step the trimmed cAC10 was convertedto the azido-modified mAb 13d through the action of His-TnGalNAcT in thepresence of 6-N3-GalNAc-UDP (commercially available from GlycoHub) as asubstrate. The preparation of the cAC10 bioconjugates is schematicallydepicted here below:

Example 9: Preparation of Compound 100

A solution of compound 99 (4.7 mg, 9.0 μmol) in DMF (200 μL) was addedto solid Val-Cit-PABC-MMAE (vc-PABC-MMAE, 10 mg, 8.1 μmol) followed byaddition of Et₃N (3.7 μL, 2.7 mg, 27 μmol). After 23 h,2′-(ethylenedioxy)bis(ethylamine) (1.3 μL, 1.3 mg, 8.9 μmol) in DMF wasadded (13 μL of 10% solution in DMF). The mixture was left for 4 h andpurified via reversed phase (C18) HPLC chromatography (30→90% MeCN (1%AcOH) in H₂O (1% AcOH). The product was obtained as a colourless film(10.7 mg, 7.1 μmol, 87%) LCMS (ESI′) calculated for C₇₄H₁₁₇N₁₂O₁₃S⁺(M+H⁺) 1509.83 found 1510.59.

Example 10: Transient Expression and Purification ofhis-TnGalNAcT(33-421)

His-TnGalNAcT(33-421) (identified by SEQ ID NO: 33) was codon optimizedand transiently expressed in CHO K1 cells by Evitria (Zurich,Switzerland) at 5 L scale. The supernatant was purified using a XK 16/20column packed with 25 mL Ni sepharose excel (GE Healthcare). Each runapproximately 1.5 L supernatant was loaded onto the column followed bywashing with at least 10 column volumes of buffer A (20 mM Tris buffer,5 mM imidazole, 500 mM NaCl, pH 7.5). Retained protein was eluted withbuffer B (20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 7.5). The bufferof the eluted fractions was exchanged to 25 mM Tris pH 8.0 using aHiPrep H26/10 desalting column (GE Healthcare). The purified protein wasconcentrated to at least 3 mg/mL using a Vivaspin Turbo 4ultrafiltration unit (Sartorius) and stored at −80° C. prior to furtheruse.

Example 11: Glycosyltransfer of the 6-N₃-GalNAc-UDP to Trimmed cAC10Under the Action of TnGalNAcT

Substrate 6-N3-GalNAc-UDP (11d) is used for the preparation of themodified biomolecule cAC10-(6-N₃-GalNAc)₂ 13d. Trimmed cAC10 (10 mg/mL),obtained by EndoSH treatment of cAC10 as described above in Example 8,was incubated with the substrate 6-N₃-GalNAc-UDP (2.5 mM, commerciallyavailable from GlycoHub) and 0.5 mg/mL His-TnGalNAcT(33-421) (5 w/w %)in 10 mM MnCl₂ and 25 mM Tris-HCl pH 8.0 at 30° C. After 3 hours theamount of His-TnGalNAcT(33-421) was increased to a final concentrationof 1 mg/mL (10 w/w %) and the reaction was incubated overnight at 30° C.Biomolecule 13d was purified from the reaction mixture on a HiTrapMabSelect SuRe 5 ml column (GE Healthcare) using an AKTA purifier-10 (GEHealthcare). The eluted IgG was immediately neutralized with 1.5 MTris-HCl pH 8.8 and dialyzed against PBS pH 7.4. Next the IgG wasconcentrated using an Amicon Ultra-0.5, Ultracel-10 Membrane (Millipore)to a concentration of 23.38 mg/mL. Mass spectral analysis of afabricator-digested sample showed three peaks of the Fc/2-fragmentbelonging to one major product (observed mass 24333 Da, approximately80% of total Fc/2 fragment), corresponding to core6-N₃-GalNAc-GlcNAc(Fuc)-substituted cAC10, and two minor products(observed masses of 24187 and 24461 Da, approximately 5 and 15% of totalFc/2 fragment), corresponding to core 6-N₃-GalNAc-GlcNAc-substitutedcAC10 and core 6-N₃-GalNAc-GlcNAc(Fuc)-substituted cAC10 with C-terminallysine.

Example 12: Conjugation of 13d with 100 to Obtain Conjugate 113

A bioconjugate according to the invention was prepared by conjugation ofcompound 100 as linker-conjugate to modified biomolecule 13d asbiomolecule. To a solution of cAC10(azide)₂ (13d) (287 μL, 6.7 mg, 23.38mg/ml in PBS pH 7.4) was added PBS pH 7.4 (133 μL) and compound 100 (27μL, 10 mM solution in DMF). The reaction was incubated at rt overnightfollowed by purification on a Superdex200 10/300 GL (GE Healthcare) onan AKTA Purifier-10 (GE Healthcare). Mass spectral analysis of thefabricator-digested sample showed one major product (observed mass 25844Da, approximately 80% of total Fc/2 fragment), corresponding to theconjugated Fc/2 fragment. RP-HPLC analysis of the reduced sampleindicated an average DAR of 1.88.

Example 13: Comparison of Trimming Efficiency of EndoS, EndoS2 andEndoSH on RNaseB at Different Concentrations

First, enzyme dilutions of the three enzymes (EndoS and EndoS2 fromGenovis, Lund, Sweden; EndoSH as obtained in Example 3) are prepared toobtain stocks solutions with 0.25 mg/mL (dil 1), 0.125 mg/mL (dil 2) and0.025 mg/mL (dil 3). Next, 12 vials were loaded with 2.5 μL RNase B (5mg/mL) followed by 0.5 μL of dilution 1-3 (dil 2 in duplo) for eachenzyme. The reactions were incubated for 30 minutes followed by additionof 36 μL water. Of these diluted solutions 6 μL was added to 6 μL samplebuffer for SDS-page analysis. Twelve samples were loaded on SDS-page gel(4 per enzyme) and run for 70 min, stained in colloidal coomassieovernight, and finally de-stained in water (see FIG. 3 for resultinggel). Conversion percentages were calculated based on scanning ofSDS-PAGE gel with regular flatbed scanner and quantification with asoftware tool (CLIQS v1.1).

TABLE 1 Percentages trimming (conversion) of RNaseB upon treatment withdifferent endoglycosidases at different enzyme concentrations. [E] mg/mLEndoS2 EndoS EndoSH 0.25 0 0 45 0.125 0 0 53 0.125 0 0 50 0.025 0 0 66

Example 14: Comparison of Trimming Efficiency of EndoS, EndoS2 andEndoSH on cAC10

cAC10 (4 mg, 20 mg/mL in Tris pH 8.0) was treated with Fabricator™(Genovis, Lund, Sweden, 4 μL, 66 U/μL) for 1 h at 37° C. Next, cleavedcAC10 was buffer exchanged to Tris pH 6.0 (50 mM, 3×) using an AmiconUltra-0.5, Ultracel-10 Membrane (Merck Millipore) to a concentration of20 mg/mL. Subsequent, three reactions containing each cAC10 (8.3 mg/mL)and an endoglycosidase (EndoS and EndoS2 from Genovis, Lund, Sweden;EndoSH as obtained in Example 3) at 0.83 μg/mL in Tris pH 6.0 50 mM werestarted. Samples of 2 μL were taken after 15 min and 35 min, dilutedwith 70 μL MiliQ and directly analysed by electrospray ionizationtime-of-flight (ESI-TOF) on a JEOL AccuTOF. Conversion percentages werecalculated based the intensities of the trimmed and untrimmed mass peaks(see FIG. 4 for the plot).

TABLE 2 Percentages trimming (conversion) of cAC10 upon treatment withdifferent endoglycosidases at different timepoints. Time EndoS2 EndoSEndoSH  0 min 0 0 0 15 min 15 24 34 35 min 36 73 73

Example 15: Comparison of Trimming Efficiency of EndoS, EndoS2 andEndoSH on High-Mannose Trastuzumab

High-mannose trastuzumab (1.3 mg, 8.8 mg/mL in Tris pH 8.0), obtainedthrough expression of trastuzumab in the presence of kifunensine, wastreated with Fabricator™ (3 μL, 66 U/μL) for 1 h at 37° C. Next, cleavedhigh-mannose trastuzumab was buffer exchanged to Tris pH 6.0 (50 mM, 3×)using an Amicon Ultra-0.5, Ultracel-10 Membrane (Merck Millipore) to aconcentration of 20 mg/mL. Three reactions were started containing eachhigh-mannose-trastuzumab (10 mg/mL) and an endoglycosidase (EndoS andEndoS2 from Genovis, Lund, Sweden; EndoSH as obtained in Example 3) at4.4 μg/mL in Tris pH 6.0 50 mM. Samples of 2 μL were taken after 30, 60and 120 min, diluted with 70 μL MiliQ and directly analysed byelectrospray ionization time-of-flight (ESI-TOF) on a JEOL AccuTOF.Conversion percentages were calculated based the intensities of thetrimmed and untrimmed mass peaks (see FIG. 5 for the plot).

TABLE 3 Percentages trimming (conversion) of high-mannose trastuzumabupon treatment with different endoglycosidases at different timepoints.Time EndoS2 EndoS EndoSH   0 min 0 0 0  30 min 14 14 27  60 min 25 14 40120 min 33 15 79

These experiments show that EndoSH It is more efficient in trimminghigh-mannose trastuzumab and cAC10 then EdnoS2, and EndoSH allows fromtrimming of other glycoproteins (e.g. RNAseB) which is not possible withEndoS2 since the activity is restricted to the N297 site. Thus, if anantibody, e.g. a monoclonal antibody, has some undesirable high-mannoseon a different N-glycosylation site, EndoSH would be able to trim thiswhereas EndoS2 cannot.

Example 16: Cloning of Fusion Proteins into Expression Vector

A pET22B-vector containing eitherEndoF3-(G₄S)₃-(His)₆-EF-(G₄S)₃-EfEndo18A (EndoF3-EfEndo18A), codingsequence EndoF3-EfEndo18A being identified by SEQ ID NO: 13; orEndoF2-(G₄S)₃-Hiss-EF-(G₄S)₃-EfEndo18A (EndoF2-EfEndo18A), codingsequence EndoF2-EfEndo18A being identified by SEQ ID NO: 14; orEndoS-(G₄S)₃-Hiss-EF-(G₄S)₃-EfEndo18A (EndoS-EfEndo18A), coding sequenceEndoS-EfEndo18A being identified by SEQ ID NO: 15; orEndoF3-(G₄S)₃-His₆-EF-(G₄S)₃-EndoF1 (EndoF3-EndoF1), coding sequenceEndoF3-EndoF1 being identified by SEQ ID NO: 16; orEndoF2-(G₄S)₃-Hiss-EF-(G₄S)₃-EndoF1 (EndoF2-EndoF1), coding sequenceEndoF2-EndoF1 being identified by SEQ ID NO: 17; orEndoS-(G₄S)₃-His₆-EF-(G₄S)₃-EndoF1 (EndoS-EndoF1), coding sequenceEndoS-EndoF1 being identified by SEQ ID NO: 18; orEndoF3-(G₄S)₃-His₆-EF-(G₄S)₃-EndoH (EndoF3-EndoH), coding sequenceEndoF3-EndoH being identified by SEQ ID NO: 19; orEndoF2-(G₄S)₃-His₆-EF-(G₄S)₃-EndoH (EndoF2-EndoH), coding sequenceEndoF2-EndoH being identified by SEQ ID NO: 20, between the NdeI-HindIIIsites was obtained from Genscript, Piscataway, USA.

The DNA sequence for the EndoF3-EfEndo18A fusion protein consists of theencoding residues 40-329 of EndoF3 fused via an N-terminal linkedglycine-serine (GS) linker to the coding residues 42-314 of EfEndo18A.The DNA sequence is identified by SEQ ID NO: 22. The glycine-serine (GS)linker comprises a -(G₄S)₃-(His)₆-EF-(G₄S)₃-format, allowing spacing ofthe two enzymes and at the same time introducing a IMAC-purificationtag.

The DNA sequence for the EndoF2-EfEndo18A fusion protein consists of theencoding residues 46-335 of EndoF2 fused via an N-terminal linkedglycine-serine (GS) linker to coding residues 42-314 of EfEndo18A. TheDNA sequence is identified by SEQ ID NO: 23. The glycine-serine (GS)linker comprises a -(G₄S)₃-(His)₆-EF-(G₄S)₃-format, allowing spacing ofthe two enzymes and at the same time introducing a IMAC-purificationtag.

The DNA sequence for the EndoS-EfEndo18A fusion protein consists of theencoding residues 48-995 of EndoS fused via an N-terminal linkedglycine-serine (GS) linker to the coding residues 42-314 of EfEndo18A.The DNA sequence is identified by SEQ ID NO: 24. The glycine-serine (GS)linker comprises a -(G₄S)₃-(His)₆-EF-(G₄S)₃-format, allowing spacing ofthe two enzymes and at the same time introducing a IMAC-purificationtag.

The DNA sequence for the EndoF3-EndoF1 fusion protein consists of theencoding residues 40-329 of EndoF3 fused via an N-terminal linkedglycine-serine (GS) linker to the coding residues 51-339 of EndoF1. TheDNA sequence is identified by SEQ ID NO: 25. The glycine-serine (GS)linker comprises a -(G₄S)₃-(His)₆-EF-(G₄S)₃-format, allowing spacing ofthe two enzymes and at the same time introducing a IMAC-purificationtag.

The DNA sequence for the EndoF2-EndoF1 fusion protein consists of theencoding residues 46-335 of EndoF2 fused via an N-terminal linkedglycine-serine (GS) linker to the coding residues 51-339 of EndoF1. TheDNA sequence is identified by SEQ ID NO: 26. The glycine-serine (GS)linker comprises a -(G₄S)₃-(His)₆-EF-(G₄S)₃-format, allowing spacing ofthe two enzymes and at the same time introducing a IMAC-purificationtag.

The DNA sequence for the EndoS-EndoF1 fusion protein consists of theencoding residues 48-995 of EndoS fused via an N-terminal linkedglycine-serine (GS) linker to the coding residues 51-339 of EndoF1. TheDNA sequence is identified by SEQ ID NO: 27. The glycine-serine (GS)linker comprises a -(G₄S)₃-(His)₆-EF-(G₄S)₃-format, allowing spacing ofthe two enzymes and at the same time introducing a IMAC-purificationtag.

The DNA sequence for the EndoF3-EndoH fusion protein consists of theencoding residues 40-329 of EndoF3 fused via an N-terminal linkedglycine-serine (GS) linker to the coding residues 41-313 of EndoH. TheDNA sequence is identified by SEQ ID NO: 28. The glycine-serine (GS)linker comprises a -(G₄S)₃-(His)₆-EF-(G₄S)₃-format, allowing spacing ofthe two enzymes and at the same time introducing a IMAC-purificationtag.

The DNA sequence for the EndoF2-EndoH fusion protein consists of theencoding residues 46-335 of EndoF2 fused via an N-terminal linkedglycine-serine (GS) linker to the coding residues 41-313 of EndoH. TheDNA sequence is identified by SEQ ID NO: 29. The glycine-serine (GS)linker comprises a -(G₄S)₃-(His)₆-EF-(G₄S)₃-format, allowing spacing ofthe two enzymes and at the same time introducing a IMAC-purificationtag.

The DNA sequence for the EndoS-EndoH fusion protein consists of theencoding residues 48-995 of EndoS fused via an N-terminal linkedglycine-serine (GS) linker to the coding residues 41-313 of EndoH. TheDNA sequence is identified by SEQ ID NO: 30. The glycine-serine (GS)linker comprises a -(G₄S)₃-(His)₆-EF-(G₄S)₃-format, allowing spacing ofthe two enzymes and at the same time introducing a IMAC-purificationtag.

The DNA sequence for the His₆-EndoS-EndoH fusion protein consists of theencoding residues 48-995 of EndoS directly fused (i.e. no (GS) linker)to the coding residues 41-313 of EndoH. The DNA sequence is identifiedby SEQ ID NO: 31. The Hiss-tag allows for purification by means ofIMAC-purification.

The DNA sequence for the encoding residues 33-421 of Hiss-TnGalNAcT witha N-terminal Hiss-Tag is identified by SEQ ID NO: 32. The Hiss-tagallows for purification by means of IMAC-purification.

Example 17: Small Scale E. coli Expression of Fusion Protein

Expression of the fusion proteins EndoF3-EndoF1 (SEQ ID NO: 16),EndoS-EndoF1 (SEQ ID NO:18), EndoF2-EndoH (SEQ ID NO:20) starts with thetransformation of the plasmid into BL21(DE3) cells. Next step is theinoculation of 50 mL culture (LB medium+ampilicin; 100 μg/ml) withBL21(DE3) cells. In case of His₆-EndoS-EndoH kanamycin (50 μg/mL) wasused. When the OD₆₀₀ reached a value of 0.5-0.7 the cultures wereinduced with 1 mM IPTG (50 μL of 1M stock solution). Expressions ofEndoF3-EndoF1 (SEQ ID NO:16) and EndoF2-EndoH (SEQ ID NO: 20) wererepeated on large scale as described in Examples 19 and 21.

Example 18: Small Scale Purification of Fusion Protein from E. coli byNiNTA

After overnight induction at 16° C. the cultures of expressionsEndoF3-EndoF1 (SEQ ID NO: 16), EndoS-EndoF1 (SEQ ID NO: 18),EndoF2-EndoH (SEQ ID NO: 20) were pelleted by centrifugation. Thepellets were re-suspended in 3-8 mL PBS and sonicated by SonopulsMini20, Bandelin (using microtip MS 2.5) at 70% (3×1 min) on ice. Afterthe sonication the cell debris was removed by centrifugation (10 min10000×g). The soluble extract was loaded onto a hand-made Ni sepharosecolumn (obtained from ThermoFisher Scientific and Ni sepharose from GEHealthcare). The column was first washed with buffer A (20 mM Trisbuffer, 5 mM imidazole, 500 mM NaCl, pH 7.5). Retained protein waseluted with buffer B (20 mM Tris, 500 mM NaCl, 250 mM imidazole, pH 7.5,5 mL). Fractions were analyzed by SDS-PAGE on polyacrylamide gels (12%).The fractions that contained purified target protein were combined andthe buffer was exchanged against TBS pH 7.5 by dialysis performedovernight at 4° C. The yields are shown in Table 4. The proteins weresnap-frozen and stored at −80° C. prior to further use.

TABLE 4 Yields for the small-scale purifications of fusion proteins fromE. coli by NiNTA. Protein Yield (mg) EndoF3-EndoF1 0.36 EndoS-EndoF1 2.7EndoF2-EndoH 0.39

Example 19: Large Scale E. coli Expression of Fusion Protein

Expression of the fusion proteins EndoF3-EndoF1 (SEQ ID NO:16),EndoF2-EndoH (SEQ ID NO: 20), EndoS-EfEndo18A (SEQ ID NO: 15),EndoF2-EndoF1 (SEQ ID NO: 17) and EndoF3-EndoH (SEQ ID NO: 19) startedwith the transformation of the plasmid into BL21(DE3) cells. Next stepwas the inoculation of 500 mL culture (LB medium+ampilicin; 100 μg/ml)with BL21(DE3) cells. When the OD600 reached 0.5-0.7 the cultures wereinduced with 1 mM IPTG (500 μL of 1M stock solution).

Example 20: Large Scale Purification of Fusion Protein from E. coli byNiNTA

After overnight induction at 16° C. the cultures of proteinsEndoS-EfEndo18A (SEQ ID NO: 15), EndoF2-EndoF1 (SEQ ID NO: 17),EndoF3-EndoH (SEQ ID NO: 19), EndoF3-EndoF1 (SEQ ID NO:16) andEndoF2-EndoH (SEQ ID NO: 20) were pelleted by centrifugation. Thepellets were re-suspended in 10 mL PBS/gram of pellet. The cellsuspension is lysed three times under pressure (20000-25000 psi) byFrench press using Emulsiflex C3, Avestin. After French press the celldebris was removed by centrifugation (20 minutes 10000×g). The solubleextract was loaded onto a HisTrap HP 5 mL column (GE Healthcare). Thecolumn was first washed with buffer A (20 mM Tris buffer, 5 mMimidazole, 500 mM NaCl, pH 7.5). Retained protein was eluted with bufferB (20 mM Tris, 500 mM NaCl, 250 mM imidazole, pH 7.5, 10 mL). Fractionswere analysed by SDS-PAGE on polyacrylamide gels (12%). The fractionsthat contained purified target protein were combined and the buffer wasexchanged against Tris pH 7.5 by dialysis performed overnight at 4° C.The yields are shown in the table 5 below. The proteins were snap-frozenand stored at −80° C. prior to further use.

TABLE 5 Yields for the large-scale purifications of fusion proteins fromE. coli by NiNTA. Protein Yield (mg) EndoS-EfEndo18A 55 EndoF2-EndoF122.1 EndoF3-EndoH 28 EndoF3-EndoF1 5.1 EndoF2-EndoH 3.5

Example 21: Purification of Fusion Protein from E. coli by SEC

For EndoF3-EndoF1 (SEQ ID NO:16), EndoF2-EndoF1 (SEQ ID NO: 17),EndoF3-EndoH (SEQ ID NO: 19) and EndoF2-EndoH (SEQ ID NO: 20) theNiNTA-purification, which is described in example 20, was followed bysize-exclusion chromatography (SEC) to isolate the monomer. A Superdex75 10/300 GL was installed on the Akta Purifier. The column was rinsedwith MilliQ (20 mL) followed by equilibration with TBS pH 7.5 (25 mL,0.8 mL/min). Approximately 1-3 mg of NiNTA-purified protein was loadedand run with 0.8 mL/min using TBS pH 7.5. The monomer protein wascollected and fractions were analysed by SDS-PAGE on polyacrylamide gels(12%) or by mass on AccuTOF. The yields are shown below in table 6. Theproteins were snap-frozen and stored at −80° C. prior to further use.

TABLE 6 Overview of the amount of NiNTA-purified endoglycosidase fusionprotein which was loaded onto the SEC-column and the yields for themonomer fraction. Protein Amount loaded (mg) Yield (mg) EndoF2-EndoF11.75 0.10 EndoF3-EndoH 0.80 0.02 EndoF3-EndoF1 2.30 0.79 EndoF2-EndoH1.40 0.12

Example 22: Cloning of Fusion Protein Hiss-EndoS-EndoH (withoutGS-Linker) into pET28B Expression Vector

A pET28B-vector containing His₆-EndoS-EndoH (His₆-EndoSH withoutGS-linker) coding sequence His₆-EndoS-EndoH being identified by SEQ IDNO: 21, between the NcoI-HindII sites was obtained from GenscriptPiscataway, USA.

The DNA sequence for the His₆-EndoSH fusion protein encodes a N-terminallinked IMAC-purification tag and a thrombin cleavage site fused to thecoding residues 48-995 of EndoS fused to the coding residues 41-313 ofEndoH.

Example 23: Small Scale E. coli Expression of Fusion ProteinHiss-EndoS-EndoH (without GS-Linker)

Expression of the fusion protein His₆-EndoS-EndoH (SEQ ID NO: 21) startswith the transformation of the plasmid into BL21(DE3) cells. Next stepis the inoculation of 50 mL culture (LB medium+kanamycin; 50 μg/ml) withBL21(DE3) cells. When the OD600 reached a value of 0.5 the culture wasinduced with 1 mM IPTG (50 μL of 1M stock solution).

Example 24: Small Scale Purification of Fusion Protein from E. coli byNiNTA

After overnight induction at 16° C. the culture of the expression inExample 23 was pelleted by centrifugation. The pellet was re-suspendedin 7 mL PBS and sonicated by Sonopuls Mini20, Bandelin (using microtipMS 2.5) at 70% (3×1 min) on ice. After the sonication the cell debriswas removed by centrifugation (10 min 10000×g). The soluble extract wasloaded onto a hand-made Ni sepharose column (obtained from ThermoFisherScientific and Ni sepharose from GE Healthcare). The column was firstwashed with buffer A (20 mM Tris buffer, 5 mM imidazole, 500 mM NaCl, pH7.5). Retained protein was eluted with buffer B (20 mM Tris, 500 mMNaCl, 250 mM imidazole, pH 7.5, 5 mL). Fractions were analysed bySDS-PAGE on polyacrylamide gels (12%). The fractions that containedpurified target protein were combined and the buffer was exchangedagainst TBS pH 7.5 by dialysis performed overnight at 4° C. The yieldafter dialysis is 9 mg. The product was snap-frozen and stored at −80°C. prior to further use.

Example 25: Comparison of Trimming Efficiency of EndoSH, EndoF3-EndoH,EndoS-EfEndo18A and EndoF2-EndoF1 on High-Mannose Trastuzumab

High-mannose trastuzumab (0.7 mL, 6.0 mg, 8.8 mg/mL in Tris pH 8.0), wastreated with Fabricator™ (9 μL, 50 U/μL) for 1 h at 37° C. Next,Fabricator™-digested high-mannose trastuzumab was divided into threeequal portions and buffer exchanged to 50 mM sodium citrate pH 4.5 with150 mM NaCl, 50 mM Tris.HCl pH 6.0 with 150 mM NaCl and 50 mM Tris.HClpH 7.5 with 150 mM NaCl, respectively. Buffer exchange was performedusing an Amicon Ultra-0.5, Ultracel-10 Membrane (Merck Millipore) andsamples were concentrated to a final concentration of 10 mg/mL. ForEndoSH (identified by SEQ ID NO: 1), EndoF3-EndoH (identified by SEQ IDNO: 19), EndoS-EfEndo18A (identified by SEQ ID NO: 15) and EndoF2-EndoF1(identified by SEQ ID NO: 17) dilution series of 10, 50 and 250 nM ineach of the above-mentioned reaction buffers were prepared. Thereactions were started by adding 2 μL of Fabricator™-digestedhigh-mannose trastuzumab (10 mg/mL) to 2 μL of the dilutedendoglycosidase fusion protein in the corresponding buffer, resulting ina final concentration of 5 mg/mL Fabricator™-digested IgG (67 μMFc/2-fragment) with 5, 25 and 125 nM endoglycosidase. The reactions wereincubated for 60 minutes at 37° C. Reactions were quenched by additionof 16 μL 1× Laemmli sample buffer without 2-mercaptoethanol followed byincubation for 5 minutes at 95° C. Samples (5 μL/sample) were loaded onSDS-page gel and run for 70 min (20 mA), stained in colloidal coomassieovernight, and finally de-stained in water. Conversion percentages werecalculated based on scanning of SDS-PAGE gel with regular flatbedscanner and quantification with a software tool (CLIQS v1.1).

TABLE 7 Percentages trimming (conversion) of Fabricator ™-digestedhigh-mannose trastuzumab upon treatment of various endoglycosidasefusion proteins at pH 4.5, 6.0 and 7.5. Enzyme EndoF3- EndoS- EndoF2- pH(nM) EndoSH EndoH EfEndo18A EndoF1 4.5 5 23* 26* 26* 26* 25 35 31 38 26*125 60 60 61 83 6.0 5 27* 25* 27* 28* 25 36 37 31 32 125 64 57 69 61 7.55 24* 26* 24* 26* 25 29* 31 31 30 125 61 53 58 58 Conversion wascalculated using the following formula: conversion (%) = 100 ×(Fc/2_(trimmed))/(Fc/2_(trimmed) + Fc/2_(glycosylated)). *Note: Resultsare difficult to interpret accurately due to background signal at theheight of untreated Fabricator ™-digested high-mannose trastuzumab. Nobackground correction was applied for conversion quantification.

Example 26: Comparison of Trimming Efficiency of EndoSH, EndoF3-EndoH,EndoS-EfEndo18A and EndoF2-EndoF1 on Trastuzumab

Trastuzumab (obtained from Epirus biopharma (Utrecht, The Netherlands);287 μL, 6.0 mg, 21 mg/mL in Tris pH 8.0), was treated with Fabricator™(9 μL, 50 U/μL) for 1 h at 37° C. Next, Fabricator™-digested trastuzumabwas divided into three equal portions and buffer exchanged to 50 mMsodium citrate pH 4.5 with 150 mM NaCl, 50 mM Tris.HCl pH 6.0 with 150mM NaCl and 50 mM Tris.HCl pH 7.5 with 150 mM NaCl, respectively. Bufferexchange was performed using an Amicon Ultra-0.5, Ultracel-10 Membrane(Merck Millipore) and samples were concentrated to a final concentrationof 10 mg/mL. For EndoSH (identified by SEQ ID NO: 1), EndoF3-EndoH(identified by SEQ ID NO: 19), EndoS-EfEndo18A (identified by SEQ ID NO:15) and EndoF2-EndoF1 (identified by SEQ ID NO: 17) dilution of 50 and250 nM in each of the above-mentioned buffers were prepared. Thereactions were started by adding 5 μL of Fabricator™-digestedtrastuzumab (10 mg/mL) to 5 μL of the diluted endoglycosidase fusionprotein in the corresponding buffer, resulting in a final concentrationof 5 mg/mL Fabricator™-digested IgG (67 μM Fc/2-fragment) with 25 and125 nM endoglycosidase. The reactions were incubated for 60 minutes at37° C. For each reaction a sample (4 μL) was taken and added to 16 μL 1×Laemmli sample buffer without 2-mercaptoethanol and incubated for 5minutes at 95° C. Samples (5 μL/sample) were loaded on SDS-page gel andrun for 70 min (20 mA), stained in colloidal coomassie overnight, andfinally de-stained in water. Conversion percentages were calculatedbased on scanning of SDS-PAGE gel with regular flatbed scanner andquantification with a software tool (CLIQS v1.1).

TABLE 8 Percentages trimming (conversion) of Fabricator ™-digestedtrastuzumab upon treatment of various endoglycosidase fusion proteins atpH 4.5, 6.0 and 7.5. Enzyme EndoF3- EndoS- EndoF2- pH (nM) EndoSH EndoHEfEndo18A EndoF1 4.5 25 29* 29* 29* 29* 125 28* 40 29* 50 6.0 25 72 28*77 28* 125 85 31 84 42 7.5 25 81 27* 82 26* 125 87 31 86 41 Conversionwas calculated using the following formula: conversion (%) = 100 ×(Fc/2_(trimmed))/(FC/2_(trimmed) + FC/2_(glycosylated)). *Note: Resultsare difficult to interpret accurately due to background signal at theheight of untreated Fabricator ™-digested trastuzumab. No backgroundcorrection was applied for conversion quantification.

Example 27: Trimming of Trastuzumab by EndoF3-EndoH

To demonstrate that complete conversion can be achieved for trimming oftrastuzumab by EndoF3-EndoH (identified by SEQ ID NO: 19),Fabricator™-digested trastuzumab (4 μL, 40 μg, 10 mg/mL in 50 mM sodiumcitrate pH 4.5 with 150 mM NaCl) was incubated with 50 mM sodium citratepH 4.5 with 150 mM NaCl (8.73 μL) and EndoF3-EndoH (7.27 μL, 4 μg, 0.55mg/mL in 50 mM sodium citrate pH 4.5 with 150 mM NaCl) for 60 minutes at37° C. Mass spectral analysis of a fabricator-digested sample showed onemain peak of the Fc/2-fragment (observed mass 24139 Da, approximately95% of total Fc/2 fragment), corresponding to coreGlcNAc(Fuc)-substituted trastuzumab.

Example 28: Trimming of Trastuzumab by EndoF2-EndoF1

To demonstrate that complete conversion can be achieved for trimming oftrastuzumab by EndoF2-EndoF1 (identified by SEQ ID NO: 17),Fabricator™-digested trastuzumab (4 μL, 40 μg, 10 mg/mL in 50 mM sodiumcitrate pH 4.5 with 150 mM NaCl) was incubated with 50 mM sodium citratepH 4.5 with 150 mM NaCl (11.06 μL) and EndoF2-EndoF1 (4.94 μL, 4 μg,0.81 mg/mL in 50 mM sodium citrate pH 4.5 with 150 mM NaCl) for 60minutes at 37° C. Mass spectral analysis of a fabricator-digested sampleshowed one main peak of the Fc/2-fragment (observed mass 24139 Da,approximately 95% of total Fc/2 fragment), corresponding to coreGlcNAc(Fuc)-substituted trastuzumab.

Example 29: Comparison of Trimming Efficiency of EndoSH, EndoF3-EndoF1,EndoS-EndoF1, EndoF2-EndoH on High-Mannose Trastuzumab

High-mannose trastuzumab (0.7 mL, 6.0 mg, 8.8 mg/mL in Tris pH 8.0), wastreated with Fabricator™ (9 μL, 50 U/μL) for 1 h at 37° C. Next,Fabricator™-digested high-mannose trastuzumab was divided into threeequal portions and buffer exchanged to 50 mM sodium citrate pH 4.5 with150 mM NaCl, 50 mM Tris.HCl pH 6.0 with 150 mM NaCl and 50 mM Tris.HClpH 7.5 with 150 mM NaCl, respectively. Buffer exchange was performedusing an Amicon Ultra-0.5, Ultracel-10 Membrane (Merck Millipore) andsamples were concentrated to a final concentration of 10 mg/mL. EndoSH(identified by SEQ ID NO: 1), EndoF3-EndoF1 (identified by SEQ ID NO:16), EndoS-EndoF1 (identified by SEQ ID NO: 18) and EndoF2-EndoH(identified by SEQ ID NO: 20) were diluted to a concentration of 250 nMin each of the above-mentioned reaction buffers. The reactions werestarted by adding 2 μL of Fabricator™-digested high-mannose trastuzumab(10 mg/mL) to 2 μL of the endoglycosidase fusion protein (250 nM) in thecorresponding buffer, resulting in a final concentration of 5 mg/mLFabricator™-digested IgG (67 μM Fc/2-fragment) with 125 nMendoglycosidase. The reactions were incubated for 60 minutes at 37° C.Reactions were quenched by addition of 16 μL 1× Laemmli sample bufferwithout 2-mercaptoethanol followed by incubation for 5 minutes at 95° C.Samples (5 μL/sample) were loaded on SDS-page gel and run for 70 min (20mA), stained in colloidal coomassie overnight, and finally de-stained inwater. Conversion percentages were calculated based on scanning ofSDS-PAGE gel with regular flatbed scanner and quantification with asoftware tool (CLIQS v1.1).

TABLE 9 Percentages trimming (conversion) of Fabricator ™-digestedhigh-mannose trastuzumab upon treatment of various endoglycosidasefusion proteins (125 nM) at pH 4.5, 6.0 and 7.5. EndoF3- EndoS- EndoF2-pH EndoSH EndoF1 EndoF1 EndoH 4.5 61 23* 60 33 6.0 69 19* 73 28* 7.5 6717* 72 25* Conversion was calculated using the following formula:conversion (%) = 100 × (Fc/2_(trimmed))/(FC/2_(trimmed) +FC/2_(glycosylated)). *Note: Results are difficult to interpretaccurately due to background signal at the height of untreatedFabricator ™-digested high-mannose trastuzumab. No background correctionwas applied for conversion quantification.

Example 30: Comparison of Trimming Efficiency of EndoSH, EndoF3-EndoHand EndoS-EfEndo18A, EndoF2-EndoF1 on Trastuzumab

Trastuzumab (obtained from Epirus biopharma (Utrecht, The Netherlands);287 μL, 6.0 mg, 21 mg/mL in Tris pH 8.0), was treated with Fabricator™(9 μL, 50 U/μL) for 1 h at 37° C. Next, Fabricator™-digested trastuzumabwas divided into three equal portions and buffer exchanged to 50 mMsodium citrate pH 4.5 with 150 mM NaCl, 50 mM Tris.HCl pH 6.0 with 150mM NaCl and 50 mM Tris.HCl pH 7.5 with 150 mM NaCl, respectively. Bufferexchange was performed using an Amicon Ultra-0.5, Ultracel-10 Membrane(Merck Millipore) and samples were concentrated to a final concentrationof 10 mg/mL. EndoSH (identified by SEQ ID NO: 1), EndoF3-EndoF1(identified by SEQ ID NO: 16), EndoS-EndoF1 (identified by SEQ ID NO:18) and EndoF2-EndoH (identified by SEQ ID NO: 20) were diluted to aconcentration of 250 nM in each of the above-mentioned reaction buffers.The reactions were started by adding 5 μL of Fabricator™-digestedtrastuzumab (10 mg/mL) to 5 μL of the endoglycosidase fusion protein(250 nM) in the corresponding buffer, resulting in a final concentrationof 5 mg/mL Fabricator™-digested IgG (67 μM Fc/2-fragment) with 125 nMendoglycosidase. The reactions were incubated for 60 minutes at 37° C.For each reaction, a sample (4 μL) was taken and added to 16 μL 1×Laemmli sample buffer without 2-mercaptoethanol and incubated for 5minutes at 95° C. Samples (5 μL/sample) were loaded on SDS-page gel andrun for 70 min (20 mA), stained in colloidal coomassie overnight, andfinally de-stained in water. Conversion percentages were calculatedbased on scanning of SDS-PAGE gel with regular flatbed scanner andquantification with a software tool (CLIQS v1.1).

TABLE 10 Percentages trimming (conversion) of trastuzumab upon treatmentwith different endoglycosidases (125 nM) at pH 4.5, 6.0 and 7.5. pHEndoSH EndoF3-EndoF1 EndoS-EndoF1 EndoF2-EndoH 4.5 22* 19* 23* 20* 6.077 16* 79 16* 7.5 80 14* 83 13* Conversion was calculated using thefollowing formula: conversion (%) = 100 ×(Fc/2_(trimmed))/(FC/2_(trimmed) + FC/2_(glycosylated)). *Note: Resultsare difficult to interpret accurately due to background signal at theheight of untreated Fabricator ™-digested trastuzumab. No backgroundcorrection was applied for conversion quantification.

Example 31: Comparison of Trimming Efficiency of EndoS-EndoH FusionProteins with and without Linker on High-Mannose Trastuzumab

High-mannose trastuzumab (0.7 mL, 6.0 mg, 8.8 mg/mL in Tris pH 8.0), wastreated with Fabricator™ (9 μL, 50 U/μL) for 1 h at 37° C. Next,Fabricator™-digested high-mannose trastuzumab was divided into threeequal portions and buffer exchanged to 50 mM sodium citrate pH 4.5 with150 mM NaCl, 50 mM Tris.HCl pH 6.0 with 150 mM NaCl and 50 mM Tris.HClpH 7.5 with 150 mM NaCl, respectively. Buffer exchange was performedusing an Amicon Ultra-0.5, Ultracel-10 Membrane (Merck Millipore) andsamples were concentrated to a final concentration of 10 mg/mL. ForEndoSH (identified by SEQ ID NO: 1) and Hiss-EndoSH (His₆-EndoS-EndoHwithout GS-linker; identified by SEQ ID NO: 21) dilution series of 10,50 and 250 nM in each of the above-mentioned reaction buffers wereprepared. The reactions were started by adding 2 μL ofFabricator™-digested high-mannose trastuzumab (10 mg/mL) to 2 μL of thediluted endoglycosidase fusion protein in the corresponding buffer,resulting in a final concentration of 5 mg/mL Fabricator™-digested IgG(67 μM Fc/2-fragment) with 5, 25 and 125 nM endoglycosidase. Thereactions were incubated for 60 minutes at 37° C. Reactions werequenched by addition of 16 μL 1× Laemmli sample buffer without2-mercaptoethanol followed by incubation for 5 minutes at 95° C. Samples(5 μL/sample) were loaded on SDS-page gel and run for 70 min (20 mA),stained in colloidal coomassie overnight, and finally de-stained inwater. Conversion percentages were calculated based on scanning ofSDS-PAGE gel with regular flatbed scanner and quantification with asoftware tool (CLIQS v1.1).

TABLE 11 Percentages trimming (conversion) of Fabricator ™-digestedhigh-mannose trastuzumab upon treatment with EndoS-EndoH fusion proteinswith and without linker at pH 4.5, 6.0 and 7.5. EndoSH His₆-EndoSH(without pH Enzyme (nM) (with GS-linker) GS-linker) 4.5 5 23* 23* 25 3531 125 60 63 6.0 5 27* 28* 25 36 42 125 64 66 7.5 5 24* 28* 25 29* 38125 61 63 Conversion was calculated using the following formula:conversion (%) = 100 × (Fc/2_(trimmed))/(FC/2_(trimmed) +FC/2_(glycosylated)). *Note: Results are difficult to interpretaccurately due to background signal at the height of untreatedFabricator ™-digested high-mannose trastuzumab. No background correctionwas applied for conversion quantification.

Example 32: Comparison of Trimming Efficiency of EndoS-EndoH FusionProteins with and without Linker on Trastuzumab

Trastuzumab (obtained from Epirus biopharma (Utrecht, The Netherlands);287 μL, 6.0 mg, 21 mg/mL in Tris pH 8.0), was treated with Fabricator™(9 μL, 50 U/μL) for 1 h at 37° C. Next, Fabricator™-digested trastuzumabwas divided into three equal portions and buffer exchanged to 50 mMsodium citrate pH 4.5 with 150 mM NaCl, 50 mM Tris.HCl pH 6.0 with 150mM NaCl and 50 mM Tris.HCl pH 7.5 with 150 mM NaCl, respectively. Bufferexchange was performed using an Amicon Ultra-0.5, Ultracel-10 Membrane(Merck Millipore) and samples were concentrated to a final concentrationof 10 mg/mL. For EndoSH (identified by SEQ ID NO: 1) and Hiss-EndoSH(EndoS-EndoH without GS-linker; identified by SEQ ID NO: 21) dilutionseries of 2, 10, 50 and 250 nM in each of the above-mentioned bufferswere prepared. The reactions were started by adding 5 μL ofFabricator™-digested trastuzumab (10 mg/mL) to 5 μL of the dilutedendoglycosidase fusion protein in the corresponding buffer, resulting ina final concentration of 5 mg/mL Fabricator™-digested IgG (67 μMFc/2-fragment) with 1, 5, 25 and 125 nM endoglycosidase. The reactionswere incubated for 60 minutes at 37° C. For each reaction, a sample (4μL) was taken and added to 16 μL 1× Laemmli sample buffer without2-mercaptoethanol and incubated for 5 minutes at 95° C. Samples (5μL/sample) were loaded on SDS-page gel and run for 70 min (20 mA),stained in colloidal coomassie overnight, and finally de-stained inwater. Conversion percentages were calculated based on scanning ofSDS-PAGE gel with regular flatbed scanner and quantification with asoftware tool (CLIQS v1.1).

TABLE 12 Percentages trimming (conversion) of Fabricator ™-digestedtrastuzumab upon treatment with EndoS-EndoH fusion proteins with andwithout linker at pH 4.5, 6.0 and 7.5. pH Enzyme (nM) EndoSH EndoSH, nolinker 4.5 1 30 28* 5 29* 29* 25 29* 29* 125 28* 29* 6.0 1 36 35 5 45 4625 72 73 125 85 83 7.5 1 34 40 5 47 51 25 81 80 125 87 84 Conversion wascalculated using the following formula: conversion (%) = 100 ×(Fc/2_(trimmed))/(FC/2_(trimmed) + FC/2_(glycosylated)). *Note: Resultsare difficult to interpret accurately due to background signal at theheight of untreated Fabricator ™-digested trastuzumab. No backgroundcorrection was applied for conversion quantification.

Example 33: Comparison of Trimming Efficiency of EndoSH, EndoF3-EndoHand EndoS-EfEndo18A, EndoF2-EndoF1 on cAC10

cAC10 (300 μL, 6.0 mg, 20.1 mg/mL in Tris pH 8.0), obtained by asdescribed above in Example 7, was treated with Fabricator™ (9 μL, 50U/μL) for 1 h at 37° C. Next, Fabricator™-digested cAC10 was dividedinto three equal portions and buffer exchanged to 50 mM sodium citratepH 4.5 with 150 mM NaCl, 50 mM Tris.HCl pH 6.0 with 150 mM NaCl and 50mM Tris.HCl pH 7.5 with 150 mM NaCl. Buffer exchange was performed usingan Amicon Ultra-0.5, Ultracel-10 Membrane (Merck Millipore) and sampleswere concentrated to a final concentration of 10 mg/mL. Example 26showed an optimal pH for the trimming of complex-type glycans of pH 7.5for EndoSH (identified by SEQ ID NO: 1), pH 4.5 for EndoF3-EndoH(identified by SEQ ID NO: 19), pH 7.5 for EndoS-EfEndo18A (identified bySEQ ID NO: 15) and pH 4.5 for EndoF2-EndoF1 (identified by SEQ ID NO:17). For each of the above mentioned fusion proteins a dilution serieswas prepared of 5, 50 and 500 nM in the reaction buffer with the optimalpH as mentioned above. The reactions were started by adding 5 μL ofFabricator™-digested cAC10 (10 mg/mL) to 5 μL of the dilutedendoglycosidase fusion protein in the corresponding buffer, resulting ina final concentration of 5 mg/mL Fabricator™-digested IgG (67 μMFc/2-fragment) with 2.5, 25 and 250 nM endoglycosidase fusion protein.The reactions were incubated for 60 minutes at 37° C. For each reaction,a sample (4 μL) was taken and added to 16 μL 1× Laemmli sample bufferwithout 2-mercaptoethanol and incubated for 5 minutes at 95° C. Samples(5 μL/sample) were loaded on SDS-page gel and run for 70 min (20 mA),stained in colloidal coomassie overnight, and finally de-stained inwater. Conversion percentages were calculated based on scanning ofSDS-PAGE gel with regular flatbed scanner and quantification with asoftware tool (CLIQS v1.1).

TABLE 13 Percentages trimming (conversion) of Fabricator ™-digestedcAC10 upon treatment of various endoglycosidase fusion proteins at anoptimal pH specific for each fusion protein. Enzyme EndoF3- EndoS-EndoF2- concentration EndoSH EndoH EfEndo18A EndoF1 (nM) (at pH 7.5) (atpH 4.5) (at pH 7.5) (at pH 4.5) 2.5 27* 70 26* 71 25 100 71 100 71 250100 70 100 73 Conversion was calculated using the following formula:conversion (%) = 100 × (Fc/2_(trimmed))/(FC/2_(trimmed) +FC/2_(glycosylated)). *Note: Results are difficult to interpretaccurately due to background signal at the height of untreatedFabricator ™-digested cAC10. No background correction was applied forconversion quantification.

Example 34: Comparison of Trimming Efficiency of EndoSH, EndoF3-EndoHand EndoS-EfEndo18A, EndoF2-EndoF1 on RNAseB

A stock solution of RNaseB (2 mg/mL) was prepared in 50 mM sodiumcitrate pH 4.5 with 150 mM NaCl and in 50 mM Tris.HCl pH 6.0 with 150 mMNaCl. Example 26 showed an optimal pH for the trimming of high-mannoseglycans of pH 6.0 for EndoSH (identified by SEQ ID NO: 1), pH 6.0 forEndoF3-EndoH (identified by SEQ ID NO: 19), pH 6.0 for EndoS-EfEndo18A(identified by SEQ ID NO: 15) and pH 4.5 for EndoF2-EndoF1 (identifiedby SEQ ID NO: 17). For each of the above mentioned fusion proteins adilution series was prepared of 10, 50 and 250 nM in the reaction bufferwith the optimal pH as mentioned above. The reactions were started byadding 5 μL of RNase B (2 mg/mL in the corresponding buffer) to 5 μL ofthe diluted endoglycosidase fusion protein in the optimal reactionbuffer as mentioned above. This results in a final concentration of 1mg/mL RNase B with 5, 25 and 125 nM endoglycosidase fusion protein. Thereactions were incubated for 60 minutes at 37° C. For each reaction, asample (4 μL) was taken and added to 16 μL 1× Laemmli sample bufferwithout 2-mercaptoethanol and incubated for 5 minutes at 95° C. Samples(5 μL/sample) were loaded on SDS-page gel and run for 70 min (20 mA),stained in colloidal coomassie overnight, and finally de-stained inwater. Conversion percentages were calculated based on scanning ofSDS-PAGE gel with regular flatbed scanner and quantification with asoftware tool (CLIQS v1.1).

TABLE 14 Percentages trimming (conversion) of RNase B upon treatment ofvarious endoglycosidase fusion proteins at the optimal pH value specificfor each fusion protein. Enzyme EndoF3- EndoS- EndoF2- concentrationEndoSH EndoH EfEndo18A EndoF1 (nM) (at pH 6.0) (at pH 6.0) (at pH 6.0)(at pH 4.5) 5 34 37 52 43 25 100 100 100 100 125 100 100 100 100Conversion was calculated using the following formula: conversion (%) =100 × (RNaseB_(trimmed))/(RNaseB_(trimmed) + RNaSeB_(glycosylated)).

Example 35: Comparison of Trimming Efficiency of EndoSH, EndoF3-EndoHand EndoS-EfEndo18A, EndoF2-EndoF1 on Fibrinogen

Fibrinogen from human plasma (commercially available from Sigma), whichcontains one glycosylation-site on the alpha-, beta- and gamma-chain,was dissolved to a final concentration of 10 mg/mL in 50 mM Tris.HCl pH6.0 with 150 mM NaCl and in 50 mM Tris.HCl pH 7.5 with 150 mM NaCl byrotating at 300 rpm at 37° C. for 15 minutes. Fibrinogen could not bedissolved in 50 mM sodium citrate pH 4.5 with 150 mM NaCl using theabove-mentioned procedure. Example 26 showed an optimal pH for thetrimming of complex-type glycans of pH 7.5 for EndoSH (identified by SEQID NO: 1), pH 4.5 for EndoF3-EndoH (identified by SEQ ID NO: 19), pH 7.5for EndoS-EfEndo18A (identified by SEQ ID NO: 15) and pH 4.5 forEndoF2-EndoF1 (identified by SEQ ID NO: 17). For EndoSH andEndoS-EfEndo18A a dilution series was prepared of 5, 50 and 500 nM in 50mM Tris.HCl pH 7.5 with 150 mM NaCl, which is the optimal reactionbuffer for these enzymes. For EndoF3-EndoH and EndoF2-EndoF1 a dilutionseries was prepared of 5, 50 and 500 nM in 50 mM Tris.HCl pH 6.0 with150 mM NaCl, which is the most optimal pH in which fibrinogen can besolubilized. The reactions were started by adding 5 μL of fibrinogen (10mg/mL) to 5 μL of the diluted endoglycosidase fusion protein in thecorresponding buffer, resulting in a final concentration of 5 mg/mLfibrinogen with 2.5, 25 and 250 nM endoglycosidase fusion protein. Thereactions were incubated for 60 minutes at 37° C. For each reaction, asample (4 μL) was taken and added to 16 μL 1× Laemmli sample buffer with2-mercaptoethanol and incubated for 5 minutes at 95° C. Samples (5μL/sample) were loaded on SDS-page gel and run for approximately 120 min(20 mA), stained in colloidal coomassie overnight, and finallyde-stained in water. Conversion percentages were calculated for thebeta- and gamma-chain based on scanning of SDS-PAGE gel with regularflatbed scanner and quantification with a software tool (CLIQS v1.1).

TABLE 15 Percentages trimming (conversion) of fibrinogen upon treatmentof various endoglycosidase fusion proteins at the optimal pH valuespecific for each fusion protein. Enzyme EndoSH EndoF3- EndoS- EndoF2-Fibrinogen concentration (at pH EndoH EfEndo18A EndoF1 chain (nM) 7.5)(at pH 6.0) (at pH 7.5) (at pH 6.0) beta-chain 2.5 9* 10* 1* 23 25 9*10* 10* 34 250 8* 8* 7* 54 gamma- 2.5 5* 7* 6* 13 chain 25 5* 4* 5* 25250 4* 6* 4* 50 Conversion was calculated separately for the beta- andgamma-chain using the following formula: conversion (%) = 100 ×(fibrinogen_(trimmed))/(fibrinogen_(trimmed) +fibrinogen_(glycosylated)). *Note: Results are difficult to interpretaccurately due to background signals at the height of untreatedfibrinogen. No background correction was applied for conversionquantification.

Example 36: Comparison of Trimming Efficiency of Fusion Proteins EndoSHand EndoF3-EndoH with Individual Proteins EndoS, EndoF3 and EndoH onTrastuzumab

Trastuzumab (obtained from Epirus biopharma (Utrecht, The Netherlands);287 μL, 6.0 mg, 21 mg/mL in Tris pH 8.0), was treated with Fabricator™(9 μL, 50 U/μL) for 1 h at 37° C. Next, Fabricator™-digested trastuzumabwas divided into three equal portions and buffer exchanged to 50 mMsodium citrate pH 4.5 with 150 mM NaCl, 50 mM Tris.HCl pH 6.0 with 150mM NaCl and 50 mM Tris.HCl pH 7.5 with 150 mM NaCl, respectively. Bufferexchange was performed using an Amicon Ultra-0.5, Ultracel-10 Membrane(Merck Millipore) and samples were concentrated to a final concentrationof 10 mg/mL. EndoSH (identified by SEQ ID NO: 1), EndoF3-EndoH(identified by SEQ ID NO: 19), EndoS (commercially available fromGenovis, Lund, Sweden), EndoF3 (commercially available fromSigma-Aldrich, EU) and EndoH (commercially available from New EnglandBiolabs, Ipswich, USA) were diluted to 50 and 500 nM in each of theabove-mentioned buffers. The reactions were started by adding 5 μL ofFabricator™-digested trastuzumab (10 mg/mL) to 5 μL of the dilutedendoglycosidases in the corresponding buffer, resulting in a finalconcentration of 5 mg/mL Fabricator™-digested IgG (67 μM Fc/2-fragment)with 25 and 250 nM endoglycosidase. The reactions were incubated for 60minutes at 37° C. For each reaction a sample (4 μL) was taken and addedto 16 μL 1× Laemmli sample buffer without 2-mercaptoethanol andincubated for 5 minutes at 95° C. Samples (5 μL/sample) were loaded onSDS-page gel and run for 70 min (20 mA), stained in colloidal coomassieovernight, and finally de-stained in water. Conversion percentages werecalculated based on scanning of SDS-PAGE gel with regular flatbedscanner and quantification with a software tool (CLIQS v1.1).

TABLE 16 Percentages trimming (conversion) of Fabricator ™-digestedtrastuzumab upon treatment of various endoglycosidases andendoglycosidase fusion proteins at pH 4.5, 6.0 and 7.5. Enzyme EndoF3-pH (nM) EndoSH EndoH EndoS EndoF3 EndoH 4.5  25  21* 21*  22* 21* 21*250  27* 50  29* 21* 19* 6.0  25  68 23*  76 24* 21* 250  83 39  82 22*19* 7.5  25  77 26*  82 15* 21* 250 100 36 100 12* 20* Conversion wascalculated using the following formula: conversion (%) = 100 ×(Fc/2_(trimmed))/(FC/2_(trimmed) + FC/2_(glycosylated)). *Note: Resultsare difficult to interpret accurately due to background signal at theheight of untreated Fabricator ™-digested trastuzumab. No backgroundcorrection was applied for conversion quantification.

Example 37: Comparison of Trimming Efficiency of Fusion Proteins EndoSHand EndoF3-EndoH with Individual Proteins EndoS, EndoF3 and EndoH onRNaseB

A stock solution of RNaseB (2 mg/mL) was prepared in 50 mM sodiumcitrate pH 4.5 with 150 mM NaCl, in 50 mM Tris.HCl pH 6.0 with 150 mMNaCl and in 50 mM Tris.HCl pH 7.5 with 150 mM NaCl. EndoSH (identifiedby SEQ ID NO: 1), EndoF3-EndoH (identified by SEQ ID NO: 19), EndoS(commercially available from Genovis, Lund, Sweden), EndoF3(commercially available from Sigma-Aldrich, EU) and EndoH (commerciallyavailable from New England Biolabs, Ipswich, USA) were diluted to aconcentration of 50 nM in each of the above-mentioned buffers. Thereactions were started by adding 5 μL of RNase B (2 mg/mL) to 5 μL ofthe diluted endoglycosidase fusion protein (50 nM) in the correspondingreaction buffer, resulting in a final concentration of 1 mg/mL RNase Band 25 nM endoglycosidase. The reactions were incubated for 60 minutesat 37° C. For each reaction, a sample (4 μL) was taken and added to 16μL 1× Laemmli sample buffer without 2-mercaptoethanol and incubated for5 minutes at 95° C. Samples (5 μL/sample) were loaded on SDS-page geland run for 70 min (20 mA), stained in colloidal coomassie overnight,and finally de-stained in water. Conversion percentages were calculatedbased on scanning of SDS-PAGE gel with regular flatbed scanner andquantification with a software tool (CLIQS v1.1).

TABLE 17 Percentages trimming (conversion) of RNase B upon treatment ofvarious endoglycosidases and endoglycosidase fusion proteins (25 nM) atpH 4.5, 6.0 and 7.5. EndoF3- pH EndoSH EndoH EndoS EndoF3 EndoH 4.5 7475 0 0 30 6.0 70 100 0 0 39 7.5 42 48 0 0 33 Conversion was calculatedusing the following formula: conversion (%) = 100 ×(RNaseB_(trimmed))/(RNaseB_(trimmed) + RNaseB_(glycosylated)).

SequencesSequence identification of fusion protein EndoS-EndoH (or EndoSH) as expressedin E coli (SEQ. ID NO: 1):    1MPSIDSLHYL SENSKKEFKE ELSKAGQESQ KVKEILAKAQ QADKQAQELA   51KMKIPEKIPM KPLHGPLYGG YFRTWHDKTS DPTEKDKVNS MGELPKEVDL  101AFIFHDWTKD YSLFWKELAT KHVPKLNKQG TRVIRTIPWR FLAGGDNSGI  151AEDTSKYPNT PEGNKALAKA IVDEYVYKYN LDGLDVDVEH DSIPKVDKKE  201DTAGVERSIQ VFEEIGKLIG PKGVDKSRLF IMDSTYMADK NPLIERGAPY  251INLLLVQVYG SQGEKGGWEP VSNRPEKTME ERWQGYSKYI RPEQYMIGFS  301FYEENAQEGN LWYDINSRKD EDKANGINTD ITGTRAERYA RWQPKTGGVK  351GGIFSYAIDR DGVAHQPKKY AKQKEFKDAT DNIFHSDYSV SKALKTVMLK  401DKSYDLIDEK DFPDKALREA VMAQVGTRKG DLERFNGTLR LDNPAIQSLE  451GLNKFKKLAQ LDLIGLSRIT KLDRSVLPAN MKPGKDTLET VLETYKKDNK  501EEPATIPPVS LKVSGLTGLK ELDLSGFDRE TLAGLDAATL TSLEKVDISG  551NKLDLAPGTE NRQIFDTMLS TISNHVGSNE QTVKFDKQKP TGHYPDTYGK  601TSLRLPVANE KVDLQSQLLF GTVTNQGTLI NSEADYKAYQ NHKIAGRSFV  651DSNYHYNNFK VSYENYTVKV TDSTLGTTTD KTLATDKEET YKVDFFSPAD  701KTKAVHTAKV IVGDEKTMMV NLAEGATVIG GSADPVNARK VFDGQLGSET  751DNISLGWDSK QSIIFKLKED GLIKHWRFFN DSARNPETTN KPIQEASLQI  801FNIKDYNLDN LLENPNKFDD EKYWITVDTY SAQGERATAF SNTLNNITSK  851YWRVVFDTKG DRYSSPVVPE LQILGYPLPN ADTIMKTVTT AKELSQQKDK  901FSQKMLDELK IKEMALETSL NSKIFDVTAI NANAGVLKDC IEKRQLLKKG  951GGGSGGGGSG GGGSHHHHHH EFGGGGSGGG GSGGGGS APA PVKQGPTSVA 1001YVEVNNNSML NVGKYTLADG GGNAFDVAVI FAANINYDTG TKTAYLHFNE 1051NVQRVLDNAV TQIRPLQQQG IKVLLSVLGN HQGAGFANFP SQQAASAFAK 1101QLSDAVAKYG LDGVDFDDEY AEYGNNGTAQ PNDSSFVHLV TALRANMPDK 1151IISLYNIGPA ASRLSYGGVD VSDKFDYAWN PYYGTWQVPG IALPKAQLSP 1201AAVEIGRTSR STVADLARRT VDEGYGVYLT YNLDGGDRTA DVSAFTRELY 1251 GSEAVRTP(linker is underlined, EndoH sequence is denoted in italics)Sequence identification of fusion protein EndoS-EndoH (or EndoSH) as expressedin CHO (SEQ. ID NO: 2):    1MPSIDSLHYL SENSKKEFKE ELSKAGQESQ KVKEILAKAQ QADKQAQELA   51KMKIPEKIPM KPLHGPLYGG YFRTWHDKTS DPTEKDKVNS MGELPKEVDL  101AFIFHDWTKD YSLFWKELAT KHVPKLNKQG TRVIRTIPWR FLAGGDNSGI  151AEDTSKYPNT PEGNKALAKA IVDEYVYKYN LDGLDVDVEH DSIPKVDKKE  201DTAGVERSIQ VFEEIGKLIG PKGVDKSRLF IMDSTYMADK NPLIERGAPY  251INLLLVQVYG SQGEKGGWEP VSNRPEKTME ERWQGYSKYI RPEQYMIGFS  301FYEENAQEGN LWYDINSRKD EDKANGINTD ITGTRAERYA RWQPKTGGVK  351GGIFSYAIDR DGVAHQPKKY AKQKEFKDAT DNIFHSDYSV SKALKTVMLK  401DKSYDLIDEK DFPDKALREA VMAQVGTRKG DLERFNGTLR LDNPAIQSLE  451GLNKFKKLAQ LDLIGLSRIT KLDRSVLPAN MKPGKDTLET VLETYKKDNK  501EEPATIPPVS LKVSGLTGLK ELDLSGFDRE TLAGLDAATL TSLEKVDISG  551NKLDLAPGTE NRQIFDTMLS TISNHVGSNE QTVKFDKQKP TGHYPDTYGK  601TSLRLPVANE KVDLQSQLLF GTVTNQGTLI NSEADYKAYQ NHKIAGRSFV  651DSNYHYNNFK VSYENYTVKV TDSTLGTTTD KTLATDKEET YKVDFFSPAD  701KTKAVHTAKV IVGDEKTMMV NLAEGATVIG GSADPVNARK VFDGQLGSET  751DNISLGWDSK QSIIFKLKED GLIKHWRFFN DSARNPETTN KPIQEASLQI  801FNIKDYNLDN LLENPNKFDD EKYWITVDTY SAQGERATAF SNTLNNITSK  851YWRVVFDTKG DRYSSPVVPE LQILGYPLPN ADTIMKTVTT AKELSQQKDK  901FSQKMLDELK IKEMALETSL NSKIFDVTAI NANAGVLKDC IEKRQLLKKG  951GGGSGGGGSG GGGSHHHHHH GGGGSGGGGS GGGGS APAPV KQGPTSVAYV 1001EVNNNSMLNV GKYTLADGGG NAFDVAVIFA ANINYDTGTK TAYLHFNENV 1051QRVLDNAVTQ IRPLQQQGIK VLLSVLGNHQ GAGFANFPSQ QAASAFAKQL 1101SDAVAKYGLD GVDFDDEYAE YGNNGTAQPN DSSFVHLVTA LRANMPDKII 1151SLYNIGPAAS RLSYGGVDVS DKFDYAWNPY YGTWQVPGIA LPRAQLSPAA 1201VEIGRTSRST VADLARRTVD EGYGVYLTYN LDGGDRTADV SAFTRELYGS 1251 EAVRTP(linker is underlined, EndoH sequence is denoted in italics)Sequence of His₆-TnGalNAcT(33-421) as expressed in E coli (SEQ. ID NO: 3):   1 MGSSHHHHHH SSGLVPRGSH MSPLRTYLYT PLYNATQPTL RNVERLAANW PKKIPSNYIE  61 DSEEYSIKNI SLSNHTTRAS VVHPPSSITE TASKLDKNMT IQDGAFAMIS PTPLLITKLM 121 DSIKSYVTTE DGVKKAEAVV TLPLCDSMPP DLGPITLNKT ELELEWVEKK FPEVEWGGRY 181 SPPNCTARHR VAIIVPYRDR QQHLAIFLNH MHPFLMKQQI EYGIFIVEQE GNKDFNRAKL 241 MNVGFVESQK LVAEGWQCFV FHDIDLLPLD TRNLYSCPRQ PRHMSASIDK LHFKLPYEDI 301 FGGVSAMTLE QFTRVNGFSN KYWGWGGEDD DMSYRLKKIN YHIARYKMSI ARYAMLDHKK 361 STPNPKRYQL LSQTSKTFQK DGLSTLEYEL VQVVQYHLYT HILVNIDERSSequence identification of fusion protein EndoF3-EfEndo18A as expressed inE coli (SEQ. ID NO: 13):    1MATALAGSNG VCIAYYITDG RNPTFKLKDI PDKVDMVILF GLKYWSLQDT   51TKLPGGTGMM GSFKSYKDLD TQIRSLQSRG IKVLQNIDDD VSWQSSKPGG  101FASAAAYGDA IKSIVIDKWK LDGISLDIEH SGAKPNPIPT FPGYAATGYN  151GWYSGSMAAT PAFLNVISEL TKYFGTTAPN NKQLQIASGI DVYAWNKIME  201NFRNNFNYIQ LQSYGANVSR TQLMMNYATG TNKIPASKMV FGAYAEGGTN  251QANDVEVAKW TPTQGAKGGM MIYTYNSNVS YANAVRDAVK NGGGGSGGGG  301SGGGGSHHHH HHEFGGGGSG GGGSGGGGS A STVTPKTVMY VEVNNHDFNN  351VGKYTLAGTN QPAFDMGIIF AANINYDTVN KKPYLYLNER VQQTLNEAET  401QIRPVQARGT KVLLSILGNH EGAGFANFPT YESADAFAAQ LEQVVNTYHL  451DGIDFDDEYA EYGKNGTPQP NNSSFIWLLQ ALRNRLGNDK LITFYNIGPA  501AANSSANPQM SSLIDYAWNP YYSTWNPPQI AGMPASRLGA SAVEVGVNQN  551LAAQYARRTK AEQYGIYLMY NLPGKDSSAY ISAATQELYG RKTNYSPTVP  601 TP(linker is underlined, EfEndo18A sequence is denoted in italics)Sequence identification of fusion protein EndoF2-EfEndo18A as expressed in E coli(SEQ. ID NO: 14):    1MAVNLSNLIA YKNSDHQISA GYYRTWRDSA TASGNLPSMR WLPDSLDMVM   51VFPDYTPPEN AYWNTLKTNY VPYLHKRGTK VIITLGDLNS ATTTGGQDSI  101GYSSWAKGIY DKWVGEYNLD GIDIDIESSP SGATLTKFVA ATKALSKYFG  151PKSGTGKTFV YDTNQNPTNF FIQTAPRYNY VFLQAYGRST TNLTTVSGLY  201APYISMKQFL PGFSFYEENG YPGNYWNDVR YPQNGTGRAY DYARWQPATG  251KKGGVFSYAI ERDAPLTSSN DNTLRAPNFR VTKDLIKIMN PGGGGSGGGG  301SGGGGSHHHH HHEFGGGGSG GGGSGGGGS A STVTPKTVMY VEVNNHDFNN  351VGKYTLAGTN QPAFDMGIIF AANINYDTVN KKPYLYLNER VQQTLNEAET  401QIRPVQARGT KVLLSILGNH EGAGFANFPT YESADAFAAQ LEQVVNTYHL  451DGIDFDDEYA EYGKNGTPQP NNSSFIWLLQ ALRNRLGNDK LITFYNIGPA  501AANSSANPQM SSLIDYAWNP YYSTWNPPQI AGMPASRLGA SAVEVGVNQN  551LAAQYAKRTK AEQYGIYLMY NLPGKDSSAY ISAATQELYG RKTNYSPTVP  601 TP(linker is underlined, EfEndo18A sequence is denoted in italics)Sequence identification of fusion protein EndoS-EfEndo18A as expressed in E coli(SEQ. ID NO: 15):    1MPSIDSLHYL SENSKKEFKE ELSKAGQESQ KVKEILAKAQ QADKQAQELA   51KMKIPEKIPM KPLHGPLYGG YFRTWHDKTS DPTEKDKVNS MGELPKEVDL  101AFIFHDWTKD YSLFWKELAT KHVPKLNKQG TRVIRTIPWR FLAGGDNSGI  151AEDTSKYPNT PEGNKALAKA IVDEYVYKYN LDGLDVDVEH DSIPKVDKKE  201DTAGVERSIQ VFEEIGKLIG PKGVDKSRLF IMDSTYMADK NPLIERGAPY  251INLLLVQVYG SQGEKGGWEP VSNRPEKTME ERWQGYSKYI RPEQYMIGFS  301FYEENAQEGN LWYDINSRKD EDKANGINTD ITGTRAERYA RWQPKTGGVK  351GGIFSYAIDR DGVAHQPKKY AKQKEFKDAT DNIFHSDYSV SKALKTVMLK  401DKSYDLIDEK DFPDKALREA VMAQVGTRKG DLERFNGTLR LDNPAIQSLE  451GLNKFKKLAQ LDLIGLSRIT KLDRSVLPAN MKPGKDTLET VLETYKKDNK  501EEPATIPPVS LKVSGLTGLK ELDLSGFDRE TLAGLDAATL TSLEKVDISG  551NKLDLAPGTE NRQIFDTMLS TISNHVGSNE QTVKFDKQKP TGHYPDTYGK  601TSLRLPVANE KVDLQSQLLF GTVTNQGTLI NSEADYKAYQ NHKIAGRSFV  651DSNYHYNNFK VSYENYTVKV TDSTLGTTTD KTLATDKEET YKVDFFSPAD  701KTKAVHTAKV IVGDEKTMMV NLAEGATVIG GSADPVNARK VFDGQLGSET  751DNISLGWDSK QSIIFKLKED GLIKHWRFFN DSARNPETTN KPIQEASLQI  801FNIKDYNLDN LLENPNKFDD EKYWITVDTY SAQGERATAF SNTLNNITSK  851YWRVVFDTKG DRYSSPVVPE LQILGYPLPN ADTIMKTVTT AKELSQQKDK  901FSQKMLDELK IKEMALETSL NSKIFDVTAI NANAGVLKDC IEKRQLLKKG  951GGGSGGGGSG GGGSHHHHHH EFGGGGSGGG GSGGGGS AST VTPKTVMYVE 1001VNNHDFNNVG KYTLAGTNQP AFDMGIIFAA NINYDTVNKK PYLYLNERVQ 1051QTLNEAETQI RPVQARGTKV LLSILGNHEG AGFANFPTYE SADAFAAQLE 1101QVVNTYHLDG IDFDDEYAEY GKNGTPQPNN SSFIWLLQAL RNRLGNDKLI 1151TFYNIGPAAA NSSANPQMSS LIDYAWNPYY STWNPPQIAG MPASRLGASA 1201VEVGVNQNLA AQYAKRTKAE QYGIYLMYNL PGKDSSAYIS AATQELYGRK 1251 TNYSPTVPTP(linker is underlined, EfEndo18A sequence is denoted in italics)Sequence identification of fusion protein EndoF3-EndoF1 as expressed in E coli(SEQ. ID NO: 16):    1MATALAGSNG VCIAYYITDG RNPTFKLKDI PDKVDMVILF GLKYWSLQDT   51TKLPGGTGMM GSFKSYKDLD TQIRSLQSRG IKVLQNIDDD VSWQSSKPGG  101FASAAAYGDA IKSIVIDKWK LDGISLDIEH SGAKPNPIPT FPGYAATGYN  151GWYSGSMAAT PAFLNVISEL TKYFGTTAPN NKQLQIASGI DVYAWNKIME  201NFRNNFNYIQ LQSYGANVSR TQLMMNYATG TNKIPASKMV FGAYAEGGTN  251QANDVEVAKW TPTQGAKGGM MIYTYNSNVS YANAVRDAVK NGGGGSGGGG  301SGGGGSHHHH HHEFGGGGSG GGGSGGGGS A VTGTTKANIK LFSFTEVNDT  351NPLNNLNFTL KNSGKPLVDM VVLFSANINY DAANDKVFVS NNPNVQHLLT  401NRAKYLKPLQ DKGIKVILSI LGNHDRSGIA NLSTARAKAF AQELKNTCDL  451YNLDGVFFDD EYSAYQTPPP SGFVTPSNNA AARLAYETKQ AMPNKLVTVY  501VYSRTSSFPT AVDGVNAGSY VDYAIHDYGG SYDLATNYPG LAKSGMVMSS  551QEFNQGRYAT AQALRNIVTK GYGGHMIFAM DPNRSNFTSG QLPALKLIAK  601ELYGDELVYS NTPYSKDW(linker is underlined, EndoF1 sequence is denoted in italics)Sequence identification of fusion protein EndoF2-EndoF1 as expressed in E coli(SEQ. ID NO: 17):    1MAVNLSNLIA YKNSDHQISA GYYRTWRDSA TASGNLPSMR WLPDSLDMVM   51VFPDYTPPEN AYWNTLKTNY VPYLHKRGTK VIITLGDLNS ATTTGGQDSI  101GYSSWAKGIY DKWVGEYNLD GIDIDIESSP SGATLTKFVA ATKALSKYFG  151PKSGTGKTFV YDTNQNPTNF FIQTAPRYNY VFLQAYGRST TNLTTVSGLY  201APYISMKQFL PGFSFYEENG YPGNYWNDVR YPQNGTGRAY DYARWQPATG  251KKGGVFSYAI ERDAPLTSSN DNTLRAPNFR VTKDLIKIMN PGGGGSGGGG  301SGGGGSHHHH HHEFGGGGSG GGGSGGGGS A VTGTTKANIK LFSFTEVNDT  351NPLNNLNFTL KNSGKPLVDM VVLFSANINY DAANDKVFVS NNPNVQHLLT  401NRAKYLKPLQ DKGIKVILSI LGNHDRSGIA NLSTARAKAF AQELKNTCDL  451YNLDGVFFDD EYSAYQTPPP SGFVTPSNNA AARLAYETKQ AMPNKLVTVY  501VYSRTSSFPT AVDGVNAGSY VDYAIHDYGG SYDLATNYPG LAKSGMVMSS  551QEFNQGRYAT AQALRNIVTK GYGGHMIFAM DPNRSNFTSG QLPALKLIAK  601ELYGDELVYS NTPYSKDW(linker is underlined, EndoF1 sequence is denoted in italics)Sequence identification of fusion protein EndoS-EndoF1 as expressed in E coli(SEQ. ID NO: 18):    1MPSIDSLHYL SENSKKEFKE ELSKAGQESQ KVKEILAKAQ QADKQAQELA   51KMKIPEKIPM KPLHGPLYGG YFRTWHDKTS DPTEKDKVNS MGELPKEVDL  101AFIFHDWTKD YSLFWKELAT KHVPKLNKQG TRVIRTIPWR FLAGGDNSGI  151AEDTSKYPNT PEGNKALAKA IVDEYVYKYN LDGLDVDVEH DSIPKVDKKE  201DTAGVERSIQ VFEEIGKLIG PKGVDKSRLF IMDSTYMADK NPLIERGAPY  251INLLLVQVYG SQGEKGGWEP VSNRPEKTME ERWQGYSKYI RPEQYMIGFS  301FYEENAQEGN LWYDINSRKD EDKANGINTD ITGTRAERYA RWQPKTGGVK  351GGIFSYAIDR DGVAHQPKKY AKQKEFKDAT DNIFHSDYSV SKALKTVMLK  401DKSYDLIDEK DFPDKALREA VMAQVGTRKG DLERFNGTLR LDNPAIQSLE  451GLNKFKKLAQ LDLIGLSRIT KLDRSVLPAN MKPGKDTLET VLETYKKDNK  501EEPATIPPVS LKVSGLTGLK ELDLSGFDRE TLAGLDAATL TSLEKVDISG  551NKLDLAPGTE NRQIFDTMLS TISNHVGSNE QTVKFDKQKP TGHYPDTYGK  601TSLRLPVANE KVDLQSQLLF GTVTNQGTLI NSEADYKAYQ NHKIAGRSFV  651DSNYHYNNFK VSYENYTVKV TDSTLGTTTD KTLATDKEET YKVDFFSPAD  701KTKAVHTAKV IVGDEKTMMV NLAEGATVIG GSADPVNARK VFDGQLGSET  751DNISLGWDSK QSIIFKLKED GLIKHWRFFN DSARNPETTN KPIQEASLQI  801FNIKDYNLDN LLENPNKFDD EKYWITVDTY SAQGERATAF SNTLNNITSK  851YWRVVFDTKG DRYSSPVVPE LQILGYPLPN ADTIMKTVTT AKELSQQKDK  901FSQKMLDELK IKEMALETSL NSKIFDVTAI NANAGVLKDC IEKRQLLKKG  951GGGSGGGGSG GGGSHHHHHH EFGGGGSGGG GSGGGGS AVT GTTKANIKLF 1001SFTEVNDTNP LNNLNFTLKN SGKPLVDMVV LFSANINYDA ANDKVFVSNN 1051PNVQHLLTNR AKYLKPLQDK GIKVILSILG NHDRSGIANL STARAKAFAQ 1101ELKNTCDLYN LDGVFFDDEY SAYQTPPPSG FVTPSNNAAA RLAYETKQAM 1151PNKLVTVYVY SRTSSFPTAV DGVNAGSYVD YAIHDYGGSY DLATNYPGLA 1201KSGMVMSSQE FNQGRYATAQ ALRNIVTKGY GGHMIFAMDP NRSNFTSGQL 1251PALKLIAKEL YGDELVYSNT PYSKDW(linker is underlined, EndoF1 sequence is denoted in italics)Sequence identification of fusion protein EndoF3-EndoH as expressed in E coli(SEQ. ID NO: 19):    1MATALAGSNG VCIAYYITDG RNPTFKLKDI PDKVDMVILF GLKYWSLQDT   51TKLPGGTGMM GSFKSYKDLD TQIRSLQSRG IKVLQNIDDD VSWQSSKPGG  101FASAAAYGDA IKSIVIDKWK LDGISLDIEH SGAKPNPIPT FPGYAATGYN  151GWYSGSMAAT PAFLNVISEL TKYFGTTAPN NKQLQIASGI DVYAWNKIME  201NFRNNFNYIQ LQSYGANVSR TQLMMNYATG TNKIPASKMV FGAYAEGGTN  251QANDVEVAKW TPTQGAKGGM MIYTYNSNVS YANAVRDAVK NGGGGSGGGG  301SGGGGSHHHH HHEFGGGGSG GGGSGGGGS A PAPVKQGPTS VAYVEVNNNS  351MLNVGKYTLA DGGGNAFDVA VIFAANINYD TGTKTAYLHF NENVQRVLDN  401AVTQIRPLQQ QGIKVLLSVL GNHQGAGFAN FPSQQAASAF AKQLSDAVAK  451YGLDGVDFDD EYAEYGNNGT AQPNDSSFVH LVTALRANMP DKIISLYNIG  501PAASRLSYGG VDVSDKFDYA WNPYYGTWQV PGIALPKAQL SPAAVEIGRT  551SRSTVADLAR RTVDEGYGVY LTYNLDGGDR TADVSAFTRE LYGSEAVRTP(linker is underlined, EndoH sequence is denoted in italics)Sequence identification of fusion protein EndoF2-EndoH as expressed in E coli(SEQ. ID NO: 20):    1MAVNLSNLIA YKNSDHQISA GYYRTWRDSA TASGNLPSMR WLPDSLDMVM   51VFPDYTPPEN AYWNTLKTNY VPYLHKRGTK VIITLGDLNS ATTTGGQDSI  101GYSSWAKGIY DKWVGEYNLD GIDIDIESSP SGATLTKFVA ATKALSKYFG  151PKSGTGKTFV YDTNQNPTNF FIQTAPRYNY VFLQAYGRST TNLTTVSGLY  201APYISMKQFL PGFSFYEENG YPGNYWNDVR YPQNGTGRAY DYARWQPATG  251KKGGVFSYAI ERDAPLTSSN DNTLRAPNFR VTKDLIKIMN PGGGGSGGGG  301SGGGGSHHHH HHEFGGGGSG GGGSGGGGS A PAPVKQGPTS VAYVEVNNNS  351MLNVGKYTLA DGGGNAFDVA VIFAANINYD TGTKTAYLHF NENVQRVLDN  401AVTQIRPLQQ QGIKVLLSVL GNHQGAGFAN FPSQQAASAF AKQLSDAVAK  451YGLDGVDFDD EYAEYGNNGT AQPNDSSFVH LVTALRANMP DKIISLYNIG  501PAASRLSYGG VDVSDKFDYA WNPYYGTWQV PGIALPRAQL SPAAVEIGRT  551SRSTVADLAR RTVDEGYGVY LTYNLDGGDR TADVSAFTRE LYGSEAVRTP(linker is underlined, EndoH sequence is denoted in italics)Sequence identification of fusion protein Hiss-EndoS-EndoH (EndoS-EndoH withoutGS-linker) as expressed in E coli (SEQ. ID NO: 21):    1MGSSHHHHHH SSGLVPRGSH MPSIDSLHYL SENSKKEFKE ELSKAGQESQ   51KVKEILAKAQ QADKQAQELA KMKIPEKIPM KPLHGPLYGG YFRTWHDKTS  101DPTEKDKVNS MGELPKEVDL AFIFHDWTKD YSLFWKELAT KHVPKLNKQG  151TRVIRTIPWR FLAGGDNSGI AEDTSKYPNT PEGNKALAKA IVDEYVYKYN  201LDGLDVDVEH DSIPKVDKKE DTAGVERSIQ VFEEIGKLIG PKGVDKSRLF  251IMDSTYMADK NPLIERGAPY INLLLVQVYG SQGEKGGWEP VSNRPEKTME  301ERWQGYSKYI RPEQYMIGFS FYEENAQEGN LWYDINSRKD EDKANGINTD  351ITGTRAERYA RWQPKTGGVK GGIFSYAIDR DGVAHQPKKY AKQKEFKDAT  401DNIFHSDYSV SKALKTVMLK DKSYDLIDEK DFPDKALREA VMAQVGTRKG  451DLERFNGTLR LDNPAIQSLE GLNKFKKLAQ LDLIGLSRIT KLDRSVLPAN  501MKPGKDTLET VLETYKKDNK EEPATIPPVS LKVSGLTGLK ELDLSGFDRE  551TLAGLDAATL TSLEKVDISG NKLDLAPGTE NRQIFDTMLS TISNHVGSNE  601QTVKFDKQKP TGHYPDTYGK TSLRLPVANE KVDLQSQLLF GTVTNQGTLI  651NSEADYKAYQ NHKIAGRSFV DSNYHYNNFK VSYENYTVKV TDSTLGTTTD  701KTLATDKEET YKVDFFSPAD KTKAVHTAKV IVGDEKTMMV NLAEGATVIG  751GSADPVNARK VFDGQLGSET DNISLGWDSK QSIIFKLKED GLIKHWRFFN  801DSARNPETTN KPIQEASLQI FNIKDYNLDN LLENPNKFDD EKYWITVDTY  851SAQGERATAF SNTLNNITSK YWRVVFDTKG DRYSSPVVPE LQILGYPLPN  901ADTIMKTVTT AKELSQQKDK FSQKMLDELK IKEMALETSL NSKIFDVTAI  951NANAGVLKDC IEKRQLLKKA PAPVKQGPTS VAYVEVNNNS MLNVGKYTLA 1001DGGGNAFDVA VIFAANINYD TGTKTAYLHF NENVQRVLDN AVTQIRPLQQ 1051QGIKVLLSVL GNHQGAGFAN FPSQQAASAF AKQLSDAVAK YGLDGVDFDD 1101EYAEYGNNGT AQPNDSSFVH LVTALRANMP DKIISLYNIG PAASRLSYGG 1151VDVSDKFDYA WNPYYGTWQV PGIALPRAQL SPAAVEIGRT SRSTVADLAR 1201RTVDEGYGVY LTYNLDGGDR TADVSAFTRE LYGSEAVRTP(N-terminal sequence including His-tag and thrombin cleavage site is underlined,EndoH sequence is in italics)Sequence identification of DNA encoding for fusion protein EndoF3-EfEndo18A asexpressed in E coli (SEQ. ID NO: 22):ATGGCTACAGCGCTGGCTGGTTCTAACGGGGTCTGCATCGCGTATTACATCACCGATGGGCGTAATCCGACGTTCAAATTGAAAGACATCCCGGATAAAGTAGACATGGTAATTCTTTTTGGTCTTAAGTATTGGTCATTGCAGGATACAACCAAATTGCCAGGGGGTACTGGTATGATGGGTTCGTTTAAATCCTACAAGGACCTGGACACCCAGATTCGTAGTCTTCAAAGCCGTGGAATCAAAGTGTTGCAGAACATTGACGACGACGTCTCATGGCAGTCCTCGAAGCCGGGTGGGTTCGCTTCCGCCGCTGCTTACGGGGATGCTATTAAGAGTATCGTAATTGATAAGTGGAAGCTGGACGGGATTAGCTTGGATATTGAGCATTCGGGGGCTAAACCCAACCCTATCCCAACTTTTCCTGGATATGCCGCGACAGGATATAATGGCTGGTATTCAGGATCTATGGCAGCCACGCCTGCCTTTCTTAATGTTATCTCAGAGCTTACTAAATACTTTGGTACAACGGCACCGAATAATAAGCAACTTCAGATTGCTTCGGGTATTGACGTATATGCCTGGAATAAAATCATGGAGAACTTTCGTAATAACTTCAACTACATCCAATTACAGTCATACGGAGCTAATGTCTCTCGTACTCAACTTATGATGAATTACGCAACGGGAACTAATAAAATTCCCGCCTCTAAAATGGTTTTCGGCGCCTACGCAGAGGGTGGCACTAACCAGGCAAATGACGTGGAGGTCGCCAAGTGGACACCTACGCAGGGCGCAAAGGGCGGTATGATGATCTATACTTACAATTCGAACGTGAGCTATGCAAATGCGGTTCGCGACGCAGTGAAAAATGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTCACCACCACCACCACCACGAATTCGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGCTTCAACCGTAACCCCTAAAACGGTTATGTACGTAGAAGTAAATAACCACGATTTCAACAATGTCGGGAAATACACTCTTGCCGGTACTAATCAGCCGGCGTTCGATATGGGTATTATTTTTGCCGCCAACATCAATTATGACACCGTCAATAAGAAACCATACCTGTACTTGAACGAGCGCGTACAGCAAACACTGAATGAAGCGGAGACGCAGATCCGTCCGGTCCAGGCACGTGGAACGAAGGTTTTGCTTTCCATCTTGGGTAATCACGAAGGCGCAGGATTTGCCAATTTTCCTACGTATGAGTCGGCGGACGCTTTCGCCGCGCAACTTGAGCAGGTTGTCAATACGTACCATTTAGACGGGATTGATTTCGATGATGAGTACGCCGAGTACGGAAAAAACGGGACCCCTCAGCCGAACAACTCATCCTTCATCTGGTTACTGCAAGCTCTTCGCAACCGTCTGGGAAATGATAAACTTATCACTTTCTACAACATTGGCCCGGCAGCCGCTAACAGCAGCGCAAACCCTCAAATGTCATCTTTGATTGACTATGCCTGGAATCCCTATTATTCGACATGGAACCCCCCACAAATTGCAGGTATGCCTGCCTCCCGCCTGGGGGCTTCTGCGGTTGAAGTGGGCGTTAACCAGAATCTTGCAGCACAGTATGCCAAGCGTACTAAGGCTGAGCAGTATGGAATCTATCTGATGTACAATCTGCCAGGAAAAGATTCTAGCGCTTATATCTCAGCAGCGACTCAGGAGCTGTATGGGCGCAAGACGAACTATAGCCCCACGGTCCCGACTCCGTGATAASequence identification of DNA encoding for fusion protein EndoF2-EfEndo18A asexpressed in E coli (SEQ. ID NO: 23):ATGGCGGTAAACCTTAGTAATCTTATCGCTTATAAAAATAGTGACCATCAGATCAGTGCGGGATATTACCGTACATGGCGTGACAGCGCCACAGCCAGTGGTAATCTTCCTAGTATGCGTTGGTTGCCAGACTCATTGGACATGGTAATGGTATTCCCAGACTATACTCCTCCGGAAAATGCGTATTGGAACACACTGAAGACTAACTACGTACCATACCTGCATAAGCGTGGCACGAAAGTTATTATCACATTGGGGGACCTTAACTCTGCAACGACCACGGGAGGGCAAGATTCTATTGGGTATTCATCGTGGGCCAAAGGAATCTATGATAAATGGGTGGGCGAGTATAATCTTGATGGAATCGATATTGACATCGAATCGTCACCGTCCGGTGCGACCTTAACGAAGTTTGTTGCGGCAACAAAAGCGTTGTCAAAGTATTTTGGACCAAAGAGTGGGACAGGCAAGACCTTTGTATACGATACCAATCAGAATCCGACTAATTTCTTTATCCAAACTGCCCCACGCTACAACTACGTATTTCTTCAAGCATACGGGCGCTCGACCACTAATCTGACGACGGTCTCTGGATTATACGCCCCCTATATTTCAATGAAACAATTTCTGCCCGGCTTCTCTTTTTACGAAGAAAACGGTTACCCAGGTAATTATTGGAATGATGTGCGTTACCCCCAGAACGGTACAGGCCGTGCCTACGACTACGCGCGCTGGCAGCCCGCCACGGGAAAAAAAGGAGGGGTGTTCAGTTATGCCATCGAGCGCGACGCCCCTCTTACATCGTCAAACGACAATACCCTGCGTGCGCCTAACTTTCGTGTAACGAAGGACTTAATCAAAATTATGAATCCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTCACCACCACCACCACCACGAATTCGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGCTTCAACCGTAACCCCTAAAACGGTTATGTACGTAGAAGTAAATAACCACGATTTCAACAATGTCGGGAAATACACTCTTGCCGGTACTAATCAGCCGGCGTTCGATATGGGTATTATTTTTGCCGCCAACATCAATTATGACACCGTCAATAAGAAACCATACCTGTACTTGAACGAGCGCGTACAGCAAACACTGAATGAAGCGGAGACGCAGATCCGTCCGGTCCAGGCACGTGGAACGAAGGTTTTGCTTTCCATCTTGGGTAATCACGAAGGCGCAGGATTTGCCAATTTTCCTACGTATGAGTCGGCGGACGCTTTCGCCGCGCAACTTGAGCAGGTTGTCAATACGTACCATTTAGACGGGATTGATTTCGATGATGAGTACGCCGAGTACGGAAAAAACGGGACCCCTCAGCCGAACAACTCATCCTTCATCTGGTTACTGCAAGCTCTTCGCAACCGTCTGGGAAATGATAAACTTATCACTTTCTACAACATTGGCCCGGCAGCCGCTAACAGCAGCGCAAACCCTCAAATGTCATCTTTGATTGACTATGCCTGGAATCCCTATTATTCGACATGGAACCCCCCACAAATTGCAGGTATGCCTGCCTCCCGCCTGGGGGCTTCTGCGGTTGAAGTGGGCGTTAACCAGAATCTTGCAGCACAGTATGCCAAGCGTACTAAGGCTGAGCAGTATGGAATCTATCTGATGTACAATCTGCCAGGAAAAGATTCTAGCGCTTATATCTCAGCAGCGACTCAGGAGCTGTATGGGCGCAAGACGAACTATAGCCCCACGGTCCCGACTCCGTGATAASequence identification of DNA encoding for fusion protein EndoS-EfEndo18A asexpressed in E coli (SEQ. ID NO: 24):ATGCCGTCAATCGATTCGCTGCATTATCTGAGCGAAAACTCTAAAAAAGAATTTAAAGAAGAACTGAGCAAAGCGGGCCAGGAATCTCAAAAAGTTAAAGAAATCCTGGCAAAAGCTCAGCAAGCCGATAAACAGGCACAAGAACTGGCTAAAATGAAAATTCCGGAAAAAATCCCGATGAAACCGCTGCATGGTCCGCTGTACGGCGGTTATTTCCGTACCTGGCACGATAAAACGTCAGACCCGACCGAAAAAGACAAAGTCAACTCGATGGGCGAACTGCCGAAAGAAGTGGATCTGGCTTTTATTTTCCATGATTGGACCAAAGACTACTCTCTGTTTTGGAAAGAACTGGCAACGAAACACGTTCCGAAACTGAACAAACAGGGTACGCGTGTCATTCGTACCATTCCGTGGCGCTTCCTGGCTGGCGGTGATAATTCAGGCATCGCGGAAGACACCTCGAAATATCCGAACACGCCGGAAGGTAATAAAGCGCTGGCCAAAGCAATCGTCGATGAATACGTGTACAAATACAATCTGGACGGCCTGGATGTGGACGTTGAACATGATTCAATTCCGAAAGTGGATAAAAAAGAAGACACCGCCGGCGTGGAACGTTCGATCCAGGTTTTTGAAGAAATTGGTAAACTGATCGGCCCGAAAGGTGTTGATAAAAGCCGTCTGTTCATCATGGATTCTACCTATATGGCCGACAAAAATCCGCTGATTGAACGCGGTGCACCGTACATCAACCTGCTGCTGGTCCAGGTGTATGGCAGCCAAGGTGAAAAAGGCGGTTGGGAACCGGTGTCTAACCGTCCGGAAAAAACCATGGAAGAACGCTGGCAGGGCTACTCAAAATATATTCGTCCGGAACAATACATGATCGGCTTTTCGTTCTATGAAGAAAACGCGCAGGAAGGTAATCTGTGGTACGATATTAATAGTCGCAAAGATGAAGACAAAGCCAACGGCATTAATACCGATATCACGGGTACCCGTGCGGAACGCTATGCCCGTTGGCAGCCGAAAACCGGCGGTGTTAAAGGCGGTATTTTTAGCTACGCGATCGATCGTGACGGTGTCGCCCATCAGCCGAAAAAATACGCAAAACAAAAAGAGTTCAAAGATGCTACCGACAACATCTTCCACAGCGATTACAGTGTCTCCAAAGCGCTGAAAACCGTGATGCTGAAAGATAAATCTTACGATCTGATCGACGAAAAAGATTTTCCGGACAAAGCGCTGCGCGAAGCCGTTATGGCACAGGTCGGCACCCGCAAAGGTGACCTGGAACGTTTTAATGGCACGCTGCGCCTGGATAACCCGGCCATTCAGAGCCTGGAAGGTCTGAATAAATTCAAAAAACTGGCACAACTGGACCTGATTGGCCTGAGCCGTATCACCAAACTGGATCGCTCTGTGCTGCCGGCCAACATGAAACCGGGTAAAGACACGCTGGAAACCGTTCTGGAAACCTACAAAAAAGATAACAAAGAAGAACCGGCAACGATCCCGCCGGTGTCTCTGAAAGTTTCCGGCCTGACCGGTCTGAAAGAACTGGATCTGAGCGGCTTTGACCGTGAAACGCTGGCAGGTCTGGATGCGGCCACGCTGACCAGTCTGGAAAAAGTTGATATTTCCGGCAATAAACTGGACCTGGCGCCGGGTACCGAAAACCGCCAGATTTTTGATACGATGCTGAGTACCATCTCCAACCATGTTGGCAGCAATGAACAGACCGTCAAATTCGACAAACAAAAACCGACGGGCCACTACCCGGATACGTATGGTAAAACCAGCCTGCGTCTGCCGGTCGCCAACGAAAAAGTGGATCTGCAGTCTCAACTGCTGTTTGGCACGGTTACCAATCAGGGTACCCTGATTAACAGCGAAGCAGATTACAAGGCTTACCAAAACCATAAAATCGCGGGTCGCTCATTTGTGGATTCGAACTACCACTACAACAACTTCAAAGTTAGTTACGAAAACTACACCGTTAAAGTCACGGATTCCACCCTGGGCACCACGACCGATAAAACGCTGGCCACCGACAAAGAAGAAACCTACAAAGTCGATTTCTTTAGCCCGGCAGACAAAACGAAAGCGGTGCATACCGCCAAAGTGATTGTTGGCGATGAAAAAACCATGATGGTGAACCTGGCTGAAGGTGCGACGGTTATCGGCGGTTCCGCAGACCCGGTTAACGCTCGCAAAGTCTTTGATGGCCAGCTGGGTAGTGAAACCGATAATATTTCCCTGGGTTGGGACTCAAAACAGTCGATTATCTTCAAACTGAAAGAAGACGGCCTGATCAAACACTGGCGTTTCTTTAACGATAGTGCCCGCAATCCGGAAACGACCAACAAACCGATTCAGGAAGCATCCCTGCAAATCTTCAACATCAAAGATTACAACCTGGACAATCTGCTGGAAAACCCGAATAAATTCGATGACGAAAAATACTGGATCACGGTGGATACCTATAGCGCGCAGGGCGAACGTGCTACGGCGTTTAGTAACACCCTGAACAATATTACGTCCAAATACTGGCGTGTGGTTTTCGATACCAAAGGTGACCGCTATAGCTCTCCGGTCGTGCCGGAACTGCAGATTCTGGGCTATCCGCTGCCGAATGCTGATACGATCATGAAAACCGTGACGACCGCGAAAGAACTGTCACAGCAAAAAGATAAATTCTCGCAGAAAATGCTGGACGAACTGAAAATTAAAGAAATGGCTCTGGAAACCAGCCTGAACAGTAAAATTTTCGATGTTACGGCGATCAATGCTAACGCTGGTGTGCTGAAAGACTGTATTGAAAAACGCCAACTGCTGAAAAAAGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTCACCACCACCACCACCACGAATTCGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGCTTCAACCGTAACCCCTAAAACGGTTATGTACGTAGAAGTAAATAACCACGATTTCAACAATGTCGGGAAATACACTCTTGCCGGTACTAATCAGCCGGCGTTCGATATGGGTATTATTTTTGCCGCCAACATCAATTATGACACCGTCAATAAGAAACCATACCTGTACTTGAACGAGCGCGTACAGCAAACACTGAATGAAGCGGAGACGCAGATCCGTCCGGTCCAGGCACGTGGAACGAAGGTTTTGCTTTCCATCTTGGGTAATCACGAAGGCGCAGGATTTGCCAATTTTCCTACGTATGAGTCGGCGGACGCTTTCGCCGCGCAACTTGAGCAGGTTGTCAATACGTACCATTTAGACGGGATTGATTTCGATGATGAGTACGCCGAGTACGGAAAAAACGGGACCCCTCAGCCGAACAACTCATCCTTCATCTGGTTACTGCAAGCTCTTCGCAACCGTCTGGGAAATGATAAACTTATCACTTTCTACAACATTGGCCCGGCAGCCGCTAACAGCAGCGCAAACCCTCAAATGTCATCTTTGATTGACTATGCCTGGAATCCCTATTATTCGACATGGAACCCCCCACAAATTGCAGGTATGCCTGCCTCCCGCCTGGGGGCTTCTGCGGTTGAAGTGGGCGTTAACCAGAATCTTGCAGCACAGTATGCCAAGCGTACTAAGGCTGAGCAGTATGGAATCTATCTGATGTACAATCTGCCAGGAAAAGATTCTAGCGCTTATATCTCAGCAGCGACTCAGGAGCTGTATGGGCGCAAGACGAACTATAGCCCCACGGTCCCGACTCCGTGATAASequence identification of DNA encoding for fusion protein EndoF3-EndoF1 asexpressed in E coli (SEQ. ID NO: 25):ATGGCTACAGCGCTGGCTGGTTCTAACGGGGTCTGCATCGCGTATTACATCACCGATGGGCGTAATCCGACGTTCAAATTGAAAGACATCCCGGATAAAGTAGACATGGTAATTCTTTTTGGTCTTAAGTATTGGTCATTGCAGGATACAACCAAATTGCCAGGGGGTACTGGTATGATGGGTTCGTTTAAATCCTACAAGGACCTGGACACCCAGATTCGTAGTCTTCAAAGCCGTGGAATCAAAGTGTTGCAGAACATTGACGACGACGTCTCATGGCAGTCCTCGAAGCCGGGTGGGTTCGCTTCCGCCGCTGCTTACGGGGATGCTATTAAGAGTATCGTAATTGATAAGTGGAAGCTGGACGGGATTAGCTTGGATATTGAGCATTCGGGGGCTAAACCCAACCCTATCCCAACTTTTCCTGGATATGCCGCGACAGGATATAATGGCTGGTATTCAGGATCTATGGCAGCCACGCCTGCCTTTCTTAATGTTATCTCAGAGCTTACTAAATACTTTGGTACAACGGCACCGAATAATAAGCAACTTCAGATTGCTTCGGGTATTGACGTATATGCCTGGAATAAAATCATGGAGAACTTTCGTAATAACTTCAACTACATCCAATTACAGTCATACGGAGCTAATGTCTCTCGTACTCAACTTATGATGAATTACGCAACGGGAACTAATAAAATTCCCGCCTCTAAAATGGTTTTCGGCGCCTACGCAGAGGGTGGCACTAACCAGGCAAATGACGTGGAGGTCGCCAAGTGGACACCTACGCAGGGCGCAAAGGGCGGTATGATGATCTATACTTACAATTCGAACGTGAGCTATGCAAATGCGGTTCGCGACGCAGTGAAAAATGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTCACCACCACCACCACCACGAATTCGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGCGGTAACCGGGACAACGAAGGCTAACATCAAACTTTTTAGTTTTACAGAGGTAAACGACACTAATCCGTTGAACAATCTGAACTTTACCTTAAAAAACTCGGGAAAACCCTTAGTAGATATGGTAGTGTTATTTTCCGCGAACATTAACTATGACGCGGCCAACGATAAGGTCTTCGTATCGAATAATCCGAACGTACAGCATCTTTTGACCAATCGTGCGAAGTACCTTAAGCCGTTACAAGACAAGGGGATCAAGGTGATTTTGTCAATCTTAGGGAACCATGATCGCTCCGGGATCGCCAATTTGAGTACGGCTCGTGCGAAGGCATTTGCTCAGGAACTGAAGAATACTTGCGATTTGTATAATTTAGACGGGGTATTCTTTGATGATGAGTACTCTGCTTACCAAACGCCACCGCCGAGCGGCTTCGTGACACCCAGTAATAACGCCGCAGCTCGCCTTGCTTATGAAACAAAGCAGGCTATGCCAAACAAGCTGGTCACGGTGTACGTCTATTCCCGCACTTCGAGTTTTCCCACAGCGGTAGACGGGGTCAACGCCGGGTCCTACGTAGACTATGCGATTCATGACTACGGTGGCTCATACGACTTGGCTACTAATTATCCGGGGTTGGCTAAGTCTGGGATGGTGATGTCTAGTCAGGAGTTTAACCAGGGCCGTTACGCGACTGCACAAGCATTGCGCAACATTGTGACCAAGGGCTATGGAGGCCACATGATCTTTGCCATGGACCCCAATCGTTCTAATTTCACGTCAGGGCAACTGCCCGCACTGAAGCTGATTGCCAAGGAGCTTTACGGGGATGAGCTTGTGTACAGCAACACTCCTTACAGTAAGGATTGGTGATAASequence identification of DNA encoding for fusion protein EndoF2-EndoF1 asexpressed in E coli (SEQ. ID NO: 26):ATGGCGGTAAACCTTAGTAATCTTATCGCTTATAAAAATAGTGACCATCAGATCAGTGCGGGATATTACCGTACATGGCGTGACAGCGCCACAGCCAGTGGTAATCTTCCTAGTATGCGTTGGTTGCCAGACTCATTGGACATGGTAATGGTATTCCCAGACTATACTCCTCCGGAAAATGCGTATTGGAACACACTGAAGACTAACTACGTACCATACCTGCATAAGCGTGGCACGAAAGTTATTATCACATTGGGGGACCTTAACTCTGCAACGACCACGGGAGGGCAAGATTCTATTGGGTATTCATCGTGGGCCAAAGGAATCTATGATAAATGGGTGGGCGAGTATAATCTTGATGGAATCGATATTGACATCGAATCGTCACCGTCCGGTGCGACCTTAACGAAGTTTGTTGCGGCAACAAAAGCGTTGTCAAAGTATTTTGGACCAAAGAGTGGGACAGGCAAGACCTTTGTATACGATACCAATCAGAATCCGACTAATTTCTTTATCCAAACTGCCCCACGCTACAACTACGTATTTCTTCAAGCATACGGGCGCTCGACCACTAATCTGACGACGGTCTCTGGATTATACGCCCCCTATATTTCAATGAAACAATTTCTGCCCGGCTTCTCTTTTTACGAAGAAAACGGTTACCCAGGTAATTATTGGAATGATGTGCGTTACCCCCAGAACGGTACAGGCCGTGCCTACGACTACGCGCGCTGGCAGCCCGCCACGGGAAAAAAAGGAGGGGTGTTCAGTTATGCCATCGAGCGCGACGCCCCTCTTACATCGTCAAACGACAATACCCTGCGTGCGCCTAACTTTCGTGTAACGAAGGACTTAATCAAAATTATGAATCCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTCACCACCACCACCACCACGAATTCGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGCGGTAACCGGGACAACGAAGGCTAACATCAAACTTTTTAGTTTTACAGAGGTAAACGACACTAATCCGTTGAACAATCTGAACTTTACCTTAAAAAACTCGGGAAAACCCTTAGTAGATATGGTAGTGTTATTTTCCGCGAACATTAACTATGACGCGGCCAACGATAAGGTCTTCGTATCGAATAATCCGAACGTACAGCATCTTTTGACCAATCGTGCGAAGTACCTTAAGCCGTTACAAGACAAGGGGATCAAGGTGATTTTGTCAATCTTAGGGAACCATGATCGCTCCGGGATCGCCAATTTGAGTACGGCTCGTGCGAAGGCATTTGCTCAGGAACTGAAGAATACTTGCGATTTGTATAATTTAGACGGGGTATTCTTTGATGATGAGTACTCTGCTTACCAAACGCCACCGCCGAGCGGCTTCGTGACACCCAGTAATAACGCCGCAGCTCGCCTTGCTTATGAAACAAAGCAGGCTATGCCAAACAAGCTGGTCACGGTGTACGTCTATTCCCGCACTTCGAGTTTTCCCACAGCGGTAGACGGGGTCAACGCCGGGTCCTACGTAGACTATGCGATTCATGACTACGGTGGCTCATACGACTTGGCTACTAATTATCCGGGGTTGGCTAAGTCTGGGATGGTGATGTCTAGTCAGGAGTTTAACCAGGGCCGTTACGCGACTGCACAAGCATTGCGCAACATTGTGACCAAGGGCTATGGAGGCCACATGATCTTTGCCATGGACCCCAATCGTTCTAATTTCACGTCAGGGCAACTGCCCGCACTGAAGCTGATTGCCAAGGAGCTTTACGGGGATGAGCTTGTGTACAGCAACACTCCTTACAGTAAGGATTGGTGATAASequence identification of DNA encoding for fusion protein EndoS-EndoF1 asexpressed in E coli (SEQ. ID NO: 27):ATGCCGTCAATCGATTCGCTGCATTATCTGAGCGAAAACTCTAAAAAAGAATTTAAAGAAGAACTGAGCAAAGCGGGCCAGGAATCTCAAAAAGTTAAAGAAATCCTGGCAAAAGCTCAGCAAGCCGATAAACAGGCACAAGAACTGGCTAAAATGAAAATTCCGGAAAAAATCCCGATGAAACCGCTGCATGGTCCGCTGTACGGCGGTTATTTCCGTACCTGGCACGATAAAACGTCAGACCCGACCGAAAAAGACAAAGTCAACTCGATGGGCGAACTGCCGAAAGAAGTGGATCTGGCTTTTATTTTCCATGATTGGACCAAAGACTACTCTCTGTTTTGGAAAGAACTGGCAACGAAACACGTTCCGAAACTGAACAAACAGGGTACGCGTGTCATTCGTACCATTCCGTGGCGCTTCCTGGCTGGCGGTGATAATTCAGGCATCGCGGAAGACACCTCGAAATATCCGAACACGCCGGAAGGTAATAAAGCGCTGGCCAAAGCAATCGTCGATGAATACGTGTACAAATACAATCTGGACGGCCTGGATGTGGACGTTGAACATGATTCAATTCCGAAAGTGGATAAAAAAGAAGACACCGCCGGCGTGGAACGTTCGATCCAGGTTTTTGAAGAAATTGGTAAACTGATCGGCCCGAAAGGTGTTGATAAAAGCCGTCTGTTCATCATGGATTCTACCTATATGGCCGACAAAAATCCGCTGATTGAACGCGGTGCACCGTACATCAACCTGCTGCTGGTCCAGGTGTATGGCAGCCAAGGTGAAAAAGGCGGTTGGGAACCGGTGTCTAACCGTCCGGAAAAAACCATGGAAGAACGCTGGCAGGGCTACTCAAAATATATTCGTCCGGAACAATACATGATCGGCTTTTCGTTCTATGAAGAAAACGCGCAGGAAGGTAATCTGTGGTACGATATTAATAGTCGCAAAGATGAAGACAAAGCCAACGGCATTAATACCGATATCACGGGTACCCGTGCGGAACGCTATGCCCGTTGGCAGCCGAAAACCGGCGGTGTTAAAGGCGGTATTTTTAGCTACGCGATCGATCGTGACGGTGTCGCCCATCAGCCGAAAAAATACGCAAAACAAAAAGAGTTCAAAGATGCTACCGACAACATCTTCCACAGCGATTACAGTGTCTCCAAAGCGCTGAAAACCGTGATGCTGAAAGATAAATCTTACGATCTGATCGACGAAAAAGATTTTCCGGACAAAGCGCTGCGCGAAGCCGTTATGGCACAGGTCGGCACCCGCAAAGGTGACCTGGAACGTTTTAATGGCACGCTGCGCCTGGATAACCCGGCCATTCAGAGCCTGGAAGGTCTGAATAAATTCAAAAAACTGGCACAACTGGACCTGATTGGCCTGAGCCGTATCACCAAACTGGATCGCTCTGTGCTGCCGGCCAACATGAAACCGGGTAAAGACACGCTGGAAACCGTTCTGGAAACCTACAAAAAAGATAACAAAGAAGAACCGGCAACGATCCCGCCGGTGTCTCTGAAAGTTTCCGGCCTGACCGGTCTGAAAGAACTGGATCTGAGCGGCTTTGACCGTGAAACGCTGGCAGGTCTGGATGCGGCCACGCTGACCAGTCTGGAAAAAGTTGATATTTCCGGCAATAAACTGGACCTGGCGCCGGGTACCGAAAACCGCCAGATTTTTGATACGATGCTGAGTACCATCTCCAACCATGTTGGCAGCAATGAACAGACCGTCAAATTCGACAAACAAAAACCGACGGGCCACTACCCGGATACGTATGGTAAAACCAGCCTGCGTCTGCCGGTCGCCAACGAAAAAGTGGATCTGCAGTCTCAACTGCTGTTTGGCACGGTTACCAATCAGGGTACCCTGATTAACAGCGAAGCAGATTACAAGGCTTACCAAAACCATAAAATCGCGGGTCGCTCATTTGTGGATTCGAACTACCACTACAACAACTTCAAAGTTAGTTACGAAAACTACACCGTTAAAGTCACGGATTCCACCCTGGGCACCACGACCGATAAAACGCTGGCCACCGACAAAGAAGAAACCTACAAAGTCGATTTCTTTAGCCCGGCAGACAAAACGAAAGCGGTGCATACCGCCAAAGTGATTGTTGGCGATGAAAAAACCATGATGGTGAACCTGGCTGAAGGTGCGACGGTTATCGGCGGTTCCGCAGACCCGGTTAACGCTCGCAAAGTCTTTGATGGCCAGCTGGGTAGTGAAACCGATAATATTTCCCTGGGTTGGGACTCAAAACAGTCGATTATCTTCAAACTGAAAGAAGACGGCCTGATCAAACACTGGCGTTTCTTTAACGATAGTGCCCGCAATCCGGAAACGACCAACAAACCGATTCAGGAAGCATCCCTGCAAATCTTCAACATCAAAGATTACAACCTGGACAATCTGCTGGAAAACCCGAATAAATTCGATGACGAAAAATACTGGATCACGGTGGATACCTATAGCGCGCAGGGCGAACGTGCTACGGCGTTTAGTAACACCCTGAACAATATTACGTCCAAATACTGGCGTGTGGTTTTCGATACCAAAGGTGACCGCTATAGCTCTCCGGTCGTGCCGGAACTGCAGATTCTGGGCTATCCGCTGCCGAATGCTGATACGATCATGAAAACCGTGACGACCGCGAAAGAACTGTCACAGCAAAAAGATAAATTCTCGCAGAAAATGCTGGACGAACTGAAAATTAAAGAAATGGCTCTGGAAACCAGCCTGAACAGTAAAATTTTCGATGTTACGGCGATCAATGCTAACGCTGGTGTGCTGAAAGACTGTATTGAAAAACGCCAACTGCTGAAAAAAGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTCACCACCACCACCACCACGAATTCGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGCGGTAACCGGGACAACGAAGGCTAACATCAAACTTTTTAGTTTTACAGAGGTAAACGACACTAATCCGTTGAACAATCTGAACTTTACCTTAAAAAACTCGGGAAAACCCTTAGTAGATATGGTAGTGTTATTTTCCGCGAACATTAACTATGACGCGGCCAACGATAAGGTCTTCGTATCGAATAATCCGAACGTACAGCATCTTTTGACCAATCGTGCGAAGTACCTTAAGCCGTTACAAGACAAGGGGATCAAGGTGATTTTGTCAATCTTAGGGAACCATGATCGCTCCGGGATCGCCAATTTGAGTACGGCTCGTGCGAAGGCATTTGCTCAGGAACTGAAGAATACTTGCGATTTGTATAATTTAGACGGGGTATTCTTTGATGATGAGTACTCTGCTTACCAAACGCCACCGCCGAGCGGCTTCGTGACACCCAGTAATAACGCCGCAGCTCGCCTTGCTTATGAAACAAAGCAGGCTATGCCAAACAAGCTGGTCACGGTGTACGTCTATTCCCGCACTTCGAGTTTTCCCACAGCGGTAGACGGGGTCAACGCCGGGTCCTACGTAGACTATGCGATTCATGACTACGGTGGCTCATACGACTTGGCTACTAATTATCCGGGGTTGGCTAAGTCTGGGATGGTGATGTCTAGTCAGGAGTTTAACCAGGGCCGTTACGCGACTGCACAAGCATTGCGCAACATTGTGACCAAGGGCTATGGAGGCCACATGATCTTTGCCATGGACCCCAATCGTTCTAATTTCACGTCAGGGCAACTGCCCGCACTGAAGCTGATTGCCAAGGAGCTTTACGGGGATGAGCTTGTGTACAGCAACACTCCTTACAGTAAGGATTGGTGATAASequence identification of DNA encoding for fusion protein EndoF3-EndoH asexpressed in E coli (SEQ. ID NO: 28):ATGGCTACAGCGCTGGCTGGTTCTAACGGGGTCTGCATCGCGTATTACATCACCGATGGGCGTAATCCGACGTTCAAATTGAAAGACATCCCGGATAAAGTAGACATGGTAATTCTTTTTGGTCTTAAGTATTGGTCATTGCAGGATACAACCAAATTGCCAGGGGGTACTGGTATGATGGGTTCGTTTAAATCCTACAAGGACCTGGACACCCAGATTCGTAGTCTTCAAAGCCGTGGAATCAAAGTGTTGCAGAACATTGACGACGACGTCTCATGGCAGTCCTCGAAGCCGGGTGGGTTCGCTTCCGCCGCTGCTTACGGGGATGCTATTAAGAGTATCGTAATTGATAAGTGGAAGCTGGACGGGATTAGCTTGGATATTGAGCATTCGGGGGCTAAACCCAACCCTATCCCAACTTTTCCTGGATATGCCGCGACAGGATATAATGGCTGGTATTCAGGATCTATGGCAGCCACGCCTGCCTTTCTTAATGTTATCTCAGAGCTTACTAAATACTTTGGTACAACGGCACCGAATAATAAGCAACTTCAGATTGCTTCGGGTATTGACGTATATGCCTGGAATAAAATCATGGAGAACTTTCGTAATAACTTCAACTACATCCAATTACAGTCATACGGAGCTAATGTCTCTCGTACTCAACTTATGATGAATTACGCAACGGGAACTAATAAAATTCCCGCCTCTAAAATGGTTTTCGGCGCCTACGCAGAGGGTGGCACTAACCAGGCAAATGACGTGGAGGTCGCCAAGTGGACACCTACGCAGGGCGCAAAGGGCGGTATGATGATCTATACTTACAATTCGAACGTGAGCTATGCAAATGCGGTTCGCGACGCAGTGAAAAATGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTCACCACCACCACCACCACGAATTCGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGCCCCGGCCCCGGTGAAGCAGGGGCCGACCTCGGTGGCCTACGTCGAGGTGAACAACAACAGCATGCTCAACGTCGGCAAGTACACCCTGGCGGACGGAGGCGGCAACGCCTTCGACGTAGCCGTGATCTTCGCGGCGAACATCAACTACGACACCGGCACGAAGACGGCCTACCTGCACTTCAACGAGAACGTGCAGCGCGTCCTTGACAACGCTGTCACGCAGATACGGCCGTTGCAGCAACAGGGCATCAAGGTCCTCCTCTCGGTGCTCGGCAACCACCAGGGCGCCGGGTTCGCGAACTTCCCCTCACAGCAGGCGGCTTCGGCGTTCGCGAAGCAGCTCTCGGACGCCGTGGCGAAGTACGGCCTCGACGGCGTCGACTTCGACGACGAATACGCCGAGTACGGCAACAACGGCACCGCGCAGCCCAACGACAGTTCGTTCGTGCACCTGGTGACGGCACTGCGCGCGAACATGCCCGACAAGATCATCAGCCTCTACAACATCGGCCCGGCCGCGTCCCGCCTGTCGTACGGCGGTGTCGACGTCTCCGACAAGTTCGACTACGCCTGGAATCCCTACTACGGCACCTGGCAGGTCCCCGGCATCGCACTGCCCAAGGCGCAGCTGTCGCCGGCGGCCGTCGAGATCGGCCGGACCTCACGGAGCACCGTCGCCGACCTCGCCCGTCGCACCGTCGACGAGGGGTACGGCGTCTATCTGACGTACAACCTCGACGGCGGCGATCGCACCGCCGACGTCTCCGCGTTCACCAGGGAGCTGTACGGCAGCGAGGCGGTCCGGACGCCGTGATAASequence identification of DNA encoding for fusion protein EndoF2-EndoH asexpressed in E coli (SEQ. ID NO: 29):ATGGCGGTAAACCTTAGTAATCTTATCGCTTATAAAAATAGTGACCATCAGATCAGTGCGGGATATTACCGTACATGGCGTGACAGCGCCACAGCCAGTGGTAATCTTCCTAGTATGCGTTGGTTGCCAGACTCATTGGACATGGTAATGGTATTCCCAGACTATACTCCTCCGGAAAATGCGTATTGGAACACACTGAAGACTAACTACGTACCATACCTGCATAAGCGTGGCACGAAAGTTATTATCACATTGGGGGACCTTAACTCTGCAACGACCACGGGAGGGCAAGATTCTATTGGGTATTCATCGTGGGCCAAAGGAATCTATGATAAATGGGTGGGCGAGTATAATCTTGATGGAATCGATATTGACATCGAATCGTCACCGTCCGGTGCGACCTTAACGAAGTTTGTTGCGGCAACAAAAGCGTTGTCAAAGTATTTTGGACCAAAGAGTGGGACAGGCAAGACCTTTGTATACGATACCAATCAGAATCCGACTAATTTCTTTATCCAAACTGCCCCACGCTACAACTACGTATTTCTTCAAGCATACGGGCGCTCGACCACTAATCTGACGACGGTCTCTGGATTATACGCCCCCTATATTTCAATGAAACAATTTCTGCCCGGCTTCTCTTTTTACGAAGAAAACGGTTACCCAGGTAATTATTGGAATGATGTGCGTTACCCCCAGAACGGTACAGGCCGTGCCTACGACTACGCGCGCTGGCAGCCCGCCACGGGAAAAAAAGGAGGGGTGTTCAGTTATGCCATCGAGCGCGACGCCCCTCTTACATCGTCAAACGACAATACCCTGCGTGCGCCTAACTTTCGTGTAACGAAGGACTTAATCAAAATTATGAATCCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTCACCACCACCACCACCACGAATTCGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGCCCCGGCCCCGGTGAAGCAGGGGCCGACCTCGGTGGCCTACGTCGAGGTGAACAACAACAGCATGCTCAACGTCGGCAAGTACACCCTGGCGGACGGAGGCGGCAACGCCTTCGACGTAGCCGTGATCTTCGCGGCGAACATCAACTACGACACCGGCACGAAGACGGCCTACCTGCACTTCAACGAGAACGTGCAGCGCGTCCTTGACAACGCTGTCACGCAGATACGGCCGTTGCAGCAACAGGGCATCAAGGTCCTCCTCTCGGTGCTCGGCAACCACCAGGGCGCCGGGTTCGCGAACTTCCCCTCACAGCAGGCGGCTTCGGCGTTCGCGAAGCAGCTCTCGGACGCCGTGGCGAAGTACGGCCTCGACGGCGTCGACTTCGACGACGAATACGCCGAGTACGGCAACAACGGCACCGCGCAGCCCAACGACAGTTCGTTCGTGCACCTGGTGACGGCACTGCGCGCGAACATGCCCGACAAGATCATCAGCCTCTACAACATCGGCCCGGCCGCGTCCCGCCTGTCGTACGGCGGTGTCGACGTCTCCGACAAGTTCGACTACGCCTGGAATCCCTACTACGGCACCTGGCAGGTCCCCGGCATCGCACTGCCCAAGGCGCAGCTGTCGCCGGCGGCCGTCGAGATCGGCCGGACCTCACGGAGCACCGTCGCCGACCTCGCCCGTCGCACCGTCGACGAGGGGTACGGCGTCTATCTGACGTACAACCTCGACGGCGGCGATCGCACCGCCGACGTCTCCGCGTTCACCAGGGAGCTGTACGGCAGCGAGGCGGTCCGGACGCCGTGATAASequence identification of DNA encoding for fusion protein EndoS-EndoH (or EndoSH)as expressed in E coli (SEQ. ID NO: 30):ATGCCGTCAATCGATTCGCTGCATTATCTGAGCGAAAACTCTAAAAAAGAATTTAAAGAAGAACTGAGCAAAGCGGGCCAGGAATCTCAAAAAGTTAAAGAAATCCTGGCAAAAGCTCAGCAAGCCGATAAACAGGCACAAGAACTGGCTAAAATGAAAATTCCGGAAAAAATCCCGATGAAACCGCTGCATGGTCCGCTGTACGGCGGTTATTTCCGTACCTGGCACGATAAAACGTCAGACCCGACCGAAAAAGACAAAGTCAACTCGATGGGCGAACTGCCGAAAGAAGTGGATCTGGCTTTTATTTTCCATGATTGGACCAAAGACTACTCTCTGTTTTGGAAAGAACTGGCAACGAAACACGTTCCGAAACTGAACAAACAGGGTACGCGTGTCATTCGTACCATTCCGTGGCGCTTCCTGGCTGGCGGTGATAATTCAGGCATCGCGGAAGACACCTCGAAATATCCGAACACGCCGGAAGGTAATAAAGCGCTGGCCAAAGCAATCGTCGATGAATACGTGTACAAATACAATCTGGACGGCCTGGATGTGGACGTTGAACATGATTCAATTCCGAAAGTGGATAAAAAAGAAGACACCGCCGGCGTGGAACGTTCGATCCAGGTTTTTGAAGAAATTGGTAAACTGATCGGCCCGAAAGGTGTTGATAAAAGCCGTCTGTTCATCATGGATTCTACCTATATGGCCGACAAAAATCCGCTGATTGAACGCGGTGCACCGTACATCAACCTGCTGCTGGTCCAGGTGTATGGCAGCCAAGGTGAAAAAGGCGGTTGGGAACCGGTGTCTAACCGTCCGGAAAAAACCATGGAAGAACGCTGGCAGGGCTACTCAAAATATATTCGTCCGGAACAATACATGATCGGCTTTTCGTTCTATGAAGAAAACGCGCAGGAAGGTAATCTGTGGTACGATATTAATAGTCGCAAAGATGAAGACAAAGCCAACGGCATTAATACCGATATCACGGGTACCCGTGCGGAACGCTATGCCCGTTGGCAGCCGAAAACCGGCGGTGTTAAAGGCGGTATTTTTAGCTACGCGATCGATCGTGACGGTGTCGCCCATCAGCCGAAAAAATACGCAAAACAAAAAGAGTTCAAAGATGCTACCGACAACATCTTCCACAGCGATTACAGTGTCTCCAAAGCGCTGAAAACCGTGATGCTGAAAGATAAATCTTACGATCTGATCGACGAAAAAGATTTTCCGGACAAAGCGCTGCGCGAAGCCGTTATGGCACAGGTCGGCACCCGCAAAGGTGACCTGGAACGTTTTAATGGCACGCTGCGCCTGGATAACCCGGCCATTCAGAGCCTGGAAGGTCTGAATAAATTCAAAAAACTGGCACAACTGGACCTGATTGGCCTGAGCCGTATCACCAAACTGGATCGCTCTGTGCTGCCGGCCAACATGAAACCGGGTAAAGACACGCTGGAAACCGTTCTGGAAACCTACAAAAAAGATAACAAAGAAGAACCGGCAACGATCCCGCCGGTGTCTCTGAAAGTTTCCGGCCTGACCGGTCTGAAAGAACTGGATCTGAGCGGCTTTGACCGTGAAACGCTGGCAGGTCTGGATGCGGCCACGCTGACCAGTCTGGAAAAAGTTGATATTTCCGGCAATAAACTGGACCTGGCGCCGGGTACCGAAAACCGCCAGATTTTTGATACGATGCTGAGTACCATCTCCAACCATGTTGGCAGCAATGAACAGACCGTCAAATTCGACAAACAAAAACCGACGGGCCACTACCCGGATACGTATGGTAAAACCAGCCTGCGTCTGCCGGTCGCCAACGAAAAAGTGGATCTGCAGTCTCAACTGCTGTTTGGCACGGTTACCAATCAGGGTACCCTGATTAACAGCGAAGCAGATTACAAGGCTTACCAAAACCATAAAATCGCGGGTCGCTCATTTGTGGATTCGAACTACCACTACAACAACTTCAAAGTTAGTTACGAAAACTACACCGTTAAAGTCACGGATTCCACCCTGGGCACCACGACCGATAAAACGCTGGCCACCGACAAAGAAGAAACCTACAAAGTCGATTTCTTTAGCCCGGCAGACAAAACGAAAGCGGTGCATACCGCCAAAGTGATTGTTGGCGATGAAAAAACCATGATGGTGAACCTGGCTGAAGGTGCGACGGTTATCGGCGGTTCCGCAGACCCGGTTAACGCTCGCAAAGTCTTTGATGGCCAGCTGGGTAGTGAAACCGATAATATTTCCCTGGGTTGGGACTCAAAACAGTCGATTATCTTCAAACTGAAAGAAGACGGCCTGATCAAACACTGGCGTTTCTTTAACGATAGTGCCCGCAATCCGGAAACGACCAACAAACCGATTCAGGAAGCATCCCTGCAAATCTTCAACATCAAAGATTACAACCTGGACAATCTGCTGGAAAACCCGAATAAATTCGATGACGAAAAATACTGGATCACGGTGGATACCTATAGCGCGCAGGGCGAACGTGCTACGGCGTTTAGTAACACCCTGAACAATATTACGTCCAAATACTGGCGTGTGGTTTTCGATACCAAAGGTGACCGCTATAGCTCTCCGGTCGTGCCGGAACTGCAGATTCTGGGCTATCCGCTGCCGAATGCTGATACGATCATGAAAACCGTGACGACCGCGAAAGAACTGTCACAGCAAAAAGATAAATTCTCGCAGAAAATGCTGGACGAACTGAAAATTAAAGAAATGGCTCTGGAAACCAGCCTGAACAGTAAAATTTTCGATGTTACGGCGATCAATGCTAACGCTGGTGTGCTGAAAGACTGTATTGAAAAACGCCAACTGCTGAAAAAAGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTCACCACCACCACCACCACGAATTCGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGCCCCGGCCCCGGTGAAGCAGGGGCCGACCTCGGTGGCCTACGTCGAGGTGAACAACAACAGCATGCTCAACGTCGGCAAGTACACCCTGGCGGACGGAGGCGGCAACGCCTTCGACGTAGCCGTGATCTTCGCGGCGAACATCAACTACGACACCGGCACGAAGACGGCCTACCTGCACTTCAACGAGAACGTGCAGCGCGTCCTTGACAACGCTGTCACGCAGATACGGCCGTTGCAGCAACAGGGCATCAAGGTCCTCCTCTCGGTGCTCGGCAACCACCAGGGCGCCGGGTTCGCGAACTTCCCCTCACAGCAGGCGGCTTCGGCGTTCGCGAAGCAGCTCTCGGACGCCGTGGCGAAGTACGGCCTCGACGGCGTCGACTTCGACGACGAATACGCCGAGTACGGCAACAACGGCACCGCGCAGCCCAACGACAGTTCGTTCGTGCACCTGGTGACGGCACTGCGCGCGAACATGCCCGACAAGATCATCAGCCTCTACAACATCGGCCCGGCCGCGTCCCGCCTGTCGTACGGCGGTGTCGACGTCTCCGACAAGTTCGACTACGCCTGGAATCCCTACTACGGCACCTGGCAGGTCCCCGGCATCGCACTGCCCAAGGCGCAGCTGTCGCCGGCGGCCGTCGAGATCGGCCGGACCTCACGGAGCACCGTCGCCGACCTCGCCCGTCGCACCGTCGACGAGGGGTACGGCGTCTATCTGACGTACAACCTCGACGGCGGCGATCGCACCGCCGACGTCTCCGCGTTCACCAGGGAGCTGTACGGCAGCGAGGCGGTCCGGACGCCGTGATAASequence identification of DNA encoding for fusion protein His₆-EndoS-EndoH (EndoS-EndoH without GS-linker) as expressed in E coli (SEQ. ID NO: 31):ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGCCGTCAATCGATTCGCTGCATTATCTGAGCGAAAACTCTAAAAAAGAATTTAAAGAAGAACTGAGCAAAGCGGGCCAGGAATCTCAAAAAGTTAAAGAAATCCTGGCAAAAGCTCAGCAAGCCGATAAACAGGCACAAGAACTGGCTAAAATGAAAATTCCGGAAAAAATCCCGATGAAACCGCTGCATGGTCCGCTGTACGGCGGTTATTTCCGTACCTGGCACGATAAAACGTCAGACCCGACCGAAAAAGACAAAGTCAACTCGATGGGCGAACTGCCGAAAGAAGTGGATCTGGCTTTTATTTTCCATGATTGGACCAAAGACTACTCTCTGTTTTGGAAAGAACTGGCAACGAAACACGTTCCGAAACTGAACAAACAGGGTACGCGTGTCATTCGTACCATTCCGTGGCGCTTCCTGGCTGGCGGTGATAATTCAGGCATCGCGGAAGACACCTCGAAATATCCGAACACGCCGGAAGGTAATAAAGCGCTGGCCAAAGCAATCGTCGATGAATACGTGTACAAATACAATCTGGACGGCCTGGATGTGGACGTTGAACATGATTCAATTCCGAAAGTGGATAAAAAAGAAGACACCGCCGGCGTGGAACGTTCGATCCAGGTTTTTGAAGAAATTGGTAAACTGATCGGCCCGAAAGGTGTTGATAAAAGCCGTCTGTTCATCATGGATTCTACCTATATGGCCGACAAAAATCCGCTGATTGAACGCGGTGCACCGTACATCAACCTGCTGCTGGTCCAGGTGTATGGCAGCCAAGGTGAAAAAGGCGGTTGGGAACCGGTGTCTAACCGTCCGGAAAAAACCATGGAAGAACGCTGGCAGGGCTACTCAAAATATATTCGTCCGGAACAATACATGATCGGCTTTTCGTTCTATGAAGAAAACGCGCAGGAAGGTAATCTGTGGTACGATATTAATAGTCGCAAAGATGAAGACAAAGCCAACGGCATTAATACCGATATCACGGGTACCCGTGCGGAACGCTATGCCCGTTGGCAGCCGAAAACCGGCGGTGTTAAAGGCGGTATTTTTAGCTACGCGATCGATCGTGACGGTGTCGCCCATCAGCCGAAAAAATACGCAAAACAAAAAGAGTTCAAAGATGCTACCGACAACATCTTCCACAGCGATTACAGTGTCTCCAAAGCGCTGAAAACCGTGATGCTGAAAGATAAATCTTACGATCTGATCGACGAAAAAGATTTTCCGGACAAAGCGCTGCGCGAAGCCGTTATGGCACAGGTCGGCACCCGCAAAGGTGACCTGGAACGTTTTAATGGCACGCTGCGCCTGGATAACCCGGCCATTCAGAGCCTGGAAGGTCTGAATAAATTCAAAAAACTGGCACAACTGGACCTGATTGGCCTGAGCCGTATCACCAAACTGGATCGCTCTGTGCTGCCGGCCAACATGAAACCGGGTAAAGACACGCTGGAAACCGTTCTGGAAACCTACAAAAAAGATAACAAAGAAGAACCGGCAACGATCCCGCCGGTGTCTCTGAAAGTTTCCGGCCTGACCGGTCTGAAAGAACTGGATCTGAGCGGCTTTGACCGTGAAACGCTGGCAGGTCTGGATGCGGCCACGCTGACCAGTCTGGAAAAAGTTGATATTTCCGGCAATAAACTGGACCTGGCGCCGGGTACCGAAAACCGCCAGATTTTTGATACGATGCTGAGTACCATCTCCAACCATGTTGGCAGCAATGAACAGACCGTCAAATTCGACAAACAAAAACCGACGGGCCACTACCCGGATACGTATGGTAAAACCAGCCTGCGTCTGCCGGTCGCCAACGAAAAAGTGGATCTGCAGTCTCAACTGCTGTTTGGCACGGTTACCAATCAGGGTACCCTGATTAACAGCGAAGCAGATTACAAGGCTTACCAAAACCATAAAATCGCGGGTCGCTCATTTGTGGATTCGAACTACCACTACAACAACTTCAAAGTTAGTTACGAAAACTACACCGTTAAAGTCACGGATTCCACCCTGGGCACCACGACCGATAAAACGCTGGCCACCGACAAAGAAGAAACCTACAAAGTCGATTTCTTTAGCCCGGCAGACAAAACGAAAGCGGTGCATACCGCCAAAGTGATTGTTGGCGATGAAAAAACCATGATGGTGAACCTGGCTGAAGGTGCGACGGTTATCGGCGGTTCCGCAGACCCGGTTAACGCTCGCAAAGTCTTTGATGGCCAGCTGGGTAGTGAAACCGATAATATTTCCCTGGGTTGGGACTCAAAACAGTCGATTATCTTCAAACTGAAAGAAGACGGCCTGATCAAACACTGGCGTTTCTTTAACGATAGTGCCCGCAATCCGGAAACGACCAACAAACCGATTCAGGAAGCATCCCTGCAAATCTTCAACATCAAAGATTACAACCTGGACAATCTGCTGGAAAACCCGAATAAATTCGATGACGAAAAATACTGGATCACGGTGGATACCTATAGCGCGCAGGGCGAACGTGCTACGGCGTTTAGTAACACCCTGAACAATATTACGTCCAAATACTGGCGTGTGGTTTTCGATACCAAAGGTGACCGCTATAGCTCTCCGGTCGTGCCGGAACTGCAGATTCTGGGCTATCCGCTGCCGAATGCTGATACGATCATGAAAACCGTGACGACCGCGAAAGAACTGTCACAGCAAAAAGATAAATTCTCGCAGAAAATGCTGGACGAACTGAAAATTAAAGAAATGGCTCTGGAAACCAGCCTGAACAGTAAAATTTTCGATGTTACGGCGATCAATGCTAACGCTGGTGTGCTGAAAGACTGTATTGAAAAACGCCAACTGCTGAAAAAAGCCCCGGCCCCGGTGAAGCAGGGGCCGACCTCGGTGGCCTACGTCGAGGTGAACAACAACAGCATGCTCAACGTCGGCAAGTACACCCTGGCGGACGGAGGCGGCAACGCCTTCGACGTAGCCGTGATCTTCGCGGCGAACATCAACTACGACACCGGCACGAAGACGGCCTACCTGCACTTCAACGAGAACGTGCAGCGCGTCCTTGACAACGCTGTCACGCAGATACGGCCGTTGCAGCAACAGGGCATCAAGGTCCTCCTCTCGGTGCTCGGCAACCACCAGGGCGCCGGGTTCGCGAACTTCCCCTCACAGCAGGCGGCTTCGGCGTTCGCGAAGCAGCTCTCGGACGCCGTGGCGAAGTACGGCCTCGACGGCGTCGACTTCGACGACGAATACGCCGAGTACGGCAACAACGGCACCGCGCAGCCCAACGACAGTTCGTTCGTGCACCTGGTGACGGCACTGCGCGCGAACATGCCCGACAAGATCATCAGCCTCTACAACATCGGCCCGGCCGCGTCCCGCCTGTCGTACGGCGGTGTCGACGTCTCCGACAAGTTCGACTACGCCTGGAATCCCTACTACGGCACCTGGCAGGTCCCCGGCATCGCACTGCCCAAGGCGCAGCTGTCGCCGGCGGCCGTCGAGATCGGCCGGACCTCACGGAGCACCGTCGCCGACCTCGCCCGTCGCACCGTCGACGAGGGGTACGGCGTCTATCTGACGTACAACCTCGACGGCGGCGATCGCACCGCCGACGTCTCCGCGTTCACCAGGGAGCTGTACGGCAGCGAGGCGGTCCGGACGCCGTGATAASequence identification of DNA encoding for His₆-TnGalNAcT(33-421) as expressed inCHO (SEQ. ID NO: 32):ATGAATTTTGGACTGAGGCTGATTTTCCTGGTGCTGACCCTGAAAGGCGTCCAGTGTCATCACCATCACCATCACTCCCCGCTTCGCACATATCTTTACACTCCATTATACAATGCCACCCAGCCCACACTCAGAAACGTCGAGAGGCTGGCAGCTAACTGGCCAAAGAAGATCCCTAGTAATTATATAGAAGATAGCGAAGAGTATAGCATCAAGAATATTTCTTTGAGCAACCACACAACTAGAGCATCTGTGGTACATCCTCCTTCCTCTATCACCGAAACGGCAAGCAAACTGGATAAGAATATGACCATCCAAGACGGCGCCTTTGCTATGATTAGCCCGACGCCCTTGCTTATCACCAAATTGATGGATAGCATCAAATCTTATGTTACTACCGAGGATGGGGTTAAGAAAGCCGAAGCCGTCGTAACTCTCCCCCTCTGTGATAGCATGCCTCCTGACCTTGGTCCTATTACTCTTAACAAAACCGAGCTCGAGCTCGAATGGGTTGAGAAAAAGTTCCCTGAGGTCGAGTGGGGTGGACGTTATAGTCCCCCCAACTGCACAGCTAGGCATCGCGTAGCAATCATAGTCCCGTACCGAGACAGACAGCAACACCTGGCAATCTTCTTAAATCACATGCACCCCTTCCTGATGAAACAGCAGATCGAATATGGCATCTTTATCGTGGAGCAGGAAGGAAACAAGGACTTTAACCGTGCGAAACTTATGAACGTCGGCTTTGTTGAAAGTCAAAAACTCGTTGCCGAGGGATGGCAGTGTTTCGTTTTTCATGACATAGACCTGCTCCCACTGGACACTAGAAACCTCTATAGCTGCCCGAGACAGCCACGCCACATGAGCGCTTCCATTGACAAACTTCACTTTAAGCTGCCTTACGAAGACATCTTCGGTGGCGTGTCAGCCATGACTCTGGAACAGTTCACCCGAGTGAATGGATTTTCAAATAAATACTGGGGATGGGGGGGAGAGGACGACGATATGAGTTATCGGCTTAAGAAAATCAACTACCATATTGCAAGATATAAAATGTCCATCGCCCGATACGCCATGTTGGACCACAAGAAGTCAACACCCAATCCTAAGCGGTACCAATTACTCTCACAGACCTCAAAGACATTCCAGAAAGACGGGCTGAGCACCCTGGAATATGAGCTGGTGCAAGTCGTTCAATATCATCTGTATACTCACATCCTGGTTAATATTGACGAGAGGTCCTGATAA (signal sequence for secretion is underlined)Sequence identification of His₆-TnGalNAcT(33-421) as expressed in CHO(SEQ. ID NO: 33):HHHHHHSPLRTYLYTPLYNATQPTLRNVERLAANWPKKIPSNYIEDSEEYSIKNISLSNHTTRASVVHPPSSITETASKLDKNMTIQDGAFAMISPTPLLITKLMDSIKSYVTTEDGVKKAEAVVTLPLCDSMPPDLGPITLNKTELELEWVEKKFPEVEWGGRYSPPNCTARHRVAIIVPYRDRQQHLAIFLNHMHPFLMKQQIEYGIFIVEQEGNKDFNRAKLMNVGFVESQKLVAEGWQCFVFHDIDLLPLDTRNLYSCPRQPRHMSASIDKLHFKLPYEDIFGGVSAMTLEQFTRVNGFSNKYWGWGGEDDDMSYRLKKINYHIARYKMSIARYAMLDHKKSTPNPKRYQLLSQTSKTFQKDGLSTLEYELVQVVQYHLYTHILVNIDERS

The invention claimed is:
 1. A fusion enzyme of structure (1):EndoX-(L)_(p)-EndoY  (1) wherein: EndoX is an endoglycosidase, EndoY isan endoglycosidase distinct from EndoX, L is a linker and p is 0 or 1,and EndoX and EndoY individually have at least 80% sequence identitywith any one of SEQ ID NO: 4-10.
 2. The fusion enzyme according to claim1, wherein EndoX and EndoY are individually selected from the groupconsisting of EfEndo18A, EndoF1, EndoF2, EndoF3, EndoH, and EndoS. 3.The fusion enzyme according to claim 1, wherein the endoglycosidasesrepresented by EndoX and EndoY have distinct endoglycosidase activity.4. The fusion enzyme according to claim 1, wherein EndoX is EndoF2,EndoF3 or EndoS.
 5. The fusion enzyme according to claim 1, whereinEndoY is EndoF1, EndoH, or EfEndo18A.
 6. The fusion enzyme according toclaim 1, having at least 50% sequence identity with any one of SEQ IDNOs: 1, 2 or 13-21.
 7. The fusion enzyme according to claim 1, whereinp=0.
 8. The fusion enzyme according to claim 1, wherein p=1 and L iscomposed of amino residues and has a length of 1 to 100 amino acidresidues.
 9. The fusion enzyme according to claim 8, wherein the linkerhas the sequence (G4S)_(n1)(H)_(r)(EF)_(s)(G₄S)_(n2), wherein n1 and n2individually are integers in the range 1-10, r is an integer in therange of 2-10 and s=0 or
 1. 10. A process for trimming a glycoprotein,comprising contacting the glycoprotein with the fusion enzyme accordingto claim
 1. 11. The process according to claim 10, wherein theglycoprotein comprises at least one high-mannose glycan and at least onecomplex glycan.
 12. The process according to claim 11, wherein theglycoprotein further comprises at least one hybrid glycan.
 13. Theprocess according to claim 10, wherein the glycoprotein is an antibody.14. The process according to claim 10, wherein the contacting isperformed at a pH which is 0.5-3 pH units different from the optimal pHof one or both of EndoX and EndoY.
 15. The process according to claim14, wherein the contacting is performed at a pH which is 1-2 pH unitsdifferent from the optimal pH of one or both of EndoX and EndoY.